Antibody drug conjugates for ablating hematopoietic stem cells

ABSTRACT

The present invention provides antibody drug conjugates, wherein an antibody or antibody fragment that specifically binds to human cKIT is linked to a drug moiety, optionally through a linker. The present invention further provides pharmaceutical compositions comprising the antibody drug conjugates; and methods of making and using such pharmaceutical compositions for ablating hematopoietic stem cells in a patient in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/437,622 filed Dec. 21, 2016, and U.S. Provisional Application No.62/520,854 filed Jun. 16, 2017, the contents of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present disclosure is directed to anti-cKIT antibody drugconjugates, and their uses for ablating hematopoietic stem cells in apatient in need thereof, e.g., a hematopoietic stem cell transplantationrecipient.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 14, 2017, isnamed PAT057400-WO-PCT_SL.txt and is 209,938 bytes in size.

BACKGROUND OF THE INVENTION

cKIT (CD117) is a single transmembrane receptor tyrosine kinase thatbinds the ligand Stem Cell Factor (SCF). SCF induces homodimerization ofcKIT which activates its tyrosine kinase activity and signals throughboth the PI3-AKT and MAPK pathways (Kindblom et al., Am J. Path. 1998152(5):1259). cKIT was initially discovered as an oncogene as atruncated form expressed by a feline retrovirus (Besmer et al., Nature1986 320:415-421). Cloning of the corresponding human gene demonstratedthat cKIT is a member of the type III class of receptor tyrosinekinases, which count among the family members, FLT3, CSF-1 receptor andPDGF receptor. cKIT is required for the development of hematopoieticcells, germ cells, mast cells and melanocytes. Hematopoietic progenitorcells, e.g., hematopoietic stem cells (HSC), in the bone marrow, expresshigh level of cKIT on cell surface. In addition, mast cells, melanocytesin the skin, and interstitial cells of Cajal in the digestive tractexpress cKIT.

Hematopoietic stem cells (HSCs) are capable of regenerating all bloodand immune cells in a transplant recipient and therefore have greattherapeutic potential. Hematopoietic stem cell transplantation is widelyused as therapies for leukemia, lymphoma, and other life-threateningdiseases. Many risks, however, are associated with such transplantation,including poor engraftment, immunological rejection, graft-versus-hostdisease (GVHD), or infection. Allogeneic hematopoietic stem celltransplantation generally requires conditioning of the recipient throughcyto-reductive treatments to prevent immunological rejection of thegraft. Current conditioning regimens are often so toxic to the host thatthey are contra-indicated for large groups of transplantation patientsand/or cannot be provided in sufficient amounts to preventgraft-versus-host disease. Thus, there is a need for improving theconditioning and transplantation methods and decreasing the risksassociated with hematopoietic stem cell transplantation and increasingits effectiveness for various disorders.

SUMMARY OF THE INVENTION

The present disclosure provides antibody drug conjugates, wherein anantibody or antibody fragment (e.g., Fab or Fab′) that specificallybinds to human cKIT is linked to a drug moiety (e.g., a cytotoxicagent), optionally through a linker. Those antibody drug conjugates canselectively deliver a cytotoxic agent to cells expressing cKIT, e.g.,hematopoietic stem cells, thereby selectively ablate those cells in apatient, e.g., a hematopoietic stem cell transplantation recipient.Preferably, the cKIT antibody drug conjugates have pharmacokineticproperties such that it will not be present and/or active in a patient'scirculation for an extended time, so they can be used for conditioninghematopoietic stem cell transplant recipients prior to hematopoieticstem cell transplantation. In some embodiments, provided herein areconjugates comprising an antibody fragment (e.g., Fab or Fab′) thatspecifically binds to cKIT, linked to a drug moiety (e.g., a cytotoxicagent), optionally through a linker. Surprisingly, the present inventorsfound that the full length anti-cKIT antibodies (e.g., full-lengthIgGs), F(ab′)₂ fragments, and toxin conjugates thereof cause mast celldegranulation, but the anti-cKIT Fab′ or Fab-toxin conjugates do notcause mast cell degranulation, even when crosslinked and/or multimerizedinto larger complexes as could be observed if a patient developed or hadpre-existing anti-drug antibodies recognizing Fab fragments. The presentdisclosure further provides pharmaceutical compositions comprising theantibody drug conjugates, and methods of making and using suchpharmaceutical compositions for ablating hematopoietic stem cells in apatient in need thereof, e.g., a hematopoietic stem cell transplantationrecipient.

In one aspect, the present disclosure is directed to a conjugate ofFormula (I):

A-(L_(B)-(D)_(n))_(y)  Formula (I);

wherein:A is an antibody fragment that specifically binds to human cKIT;LB is a linker;D is a cytotoxic agent;n is an integer from 1 to 10, and y is an integer from 1 to 10.

In one aspect, the present disclosure is directed to a conjugate ofhaving the structure of Formula (C):

wherein A, L₂₀, y and R², are as defined herein.

In one aspect, the present disclosure is directed to a conjugate ofhaving the structure of Formula (D):

wherein A, L₃₀, y, R¹ and R², are as defined herein.

In one aspect, the present disclosure is directed to a conjugate ofhaving the structure of Formula (E):

wherein A, L₄₀, y, X, R⁵ and R⁶, are as defined herein.

In another aspect, provided herein are antibodies and antibody fragments(e.g., Fab or Fab′) that specifically bind to human cKIT. Such anti-cKITantibodies and antibody fragments (e.g., Fab or Fab′) can be used in anyof the conjugates described herein.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT is an antibody or antibodyfragment (e.g., Fab or Fab′) that specifically binds to theextracellular domain of human cKIT (SEQ ID NO: 112).

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT is an antibody or antibodyfragment (e.g., Fab or Fab′) that specifically binds to an epitope indomains 1-3 of human cKIT (SEQ ID NO: 113).

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT is an antibody or antibodyfragment (e.g., Fab or Fab′) described in Table 1.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 1, a HCDR2 of SEQ ID NO: 2; a HCDR3 of SEQ ID NO: 3; a LCDR1 of SEQID NO: 16; a LCDR2 of SEQ ID NO: 17; and a LCDR3 of SEQ ID NO: 18.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 4, a HCDR2 of SEQ ID NO: 5; a HCDR3 of SEQ ID NO: 3; a LCDR1 of SEQID NO:19; a LCDR2 of SEQ ID NO: 20; and a LCDR3 of SEQ ID NO: 21.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 6, a HCDR2 of SEQ ID NO: 2; a HCDR3 of SEQ ID NO: 3; a LCDR1 of SEQID NO:16; a LCDR2 of SEQ ID NO: 17; and a LCDR3 of SEQ ID NO: 18.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 7, a HCDR2 of SEQ ID NO: 8; a HCDR3 of SEQ ID NO: 9; a LCDR1 of SEQID NO: 22; a LCDR2 of SEQ ID NO: 20; and a LCDR3 of SEQ ID NO: 18.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 27, a HCDR2 of SEQ ID NO: 28; a HCDR3 of SEQ ID NO: 29; a LCDR1 ofSEQ ID NO: 42; a LCDR2 of SEQ ID NO: 17; and a LCDR3 of SEQ ID NO: 43.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 30, a HCDR2 of SEQ ID NO: 31; a HCDR3 of SEQ ID NO: 29; a LCDR1 ofSEQ ID NO: 44; a LCDR2 of SEQ ID NO: 20; and a LCDR3 of SEQ ID NO: 45.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 32, a HCDR2 of SEQ ID NO: 28; a HCDR3 of SEQ ID NO: 29; a LCDR1 ofSEQ ID NO: 42; a LCDR2 of SEQ ID NO: 17; and a LCDR3 of SEQ ID NO: 43.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 33, a HCDR2 of SEQ ID NO: 34; a HCDR3 of SEQ ID NO: 35; a LCDR1 ofSEQ ID NO: 46; a LCDR2 of SEQ ID NO: 20; and a LCDR3 of SEQ ID NO: 43.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 1, a HCDR2 of SEQ ID NO: 51; a HCDR3 of SEQ ID NO: 3; a LCDR1 of SEQID NO:16; a LCDR2 of SEQ ID NO: 17; and a LCDR3 of SEQ ID NO: 18.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 4, a HCDR2 of SEQ ID NO: 52; a HCDR3 of SEQ ID NO: 3; a LCDR1 of SEQID NO:19; a LCDR2 of SEQ ID NO: 20; and a LCDR3 of SEQ ID NO: 21.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 6, a HCDR2 of SEQ ID NO: 51; a HCDR3 of SEQ ID NO: 3; a LCDR1 of SEQID NO:16; a LCDR2 of SEQ ID NO: 17; and a LCDR3 of SEQ ID NO: 18.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 7, a HCDR2 of SEQ ID NO: 53; a HCDR3 of SEQ ID NO: 9; a LCDR1 of SEQID NO: 22; a LCDR2 of SEQ ID NO: 20; and a LCDR3 of SEQ ID NO: 18.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 60, a HCDR2 of SEQ ID NO: 61; a HCDR3 of SEQ ID NO: 62; a LCDR1 ofSEQ ID NO: 75; a LCDR2 of SEQ ID NO: 76; and a LCDR3 of SEQ ID NO: 77.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 63, a HCDR2 of SEQ ID NO: 64; a HCDR3 of SEQ ID NO: 62; a LCDR1 ofSEQ ID NO: 78; a LCDR2 of SEQ ID NO: 79; and a LCDR3 of SEQ ID NO: 80.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 65, a HCDR2 of SEQ ID NO: 61; a HCDR3 of SEQ ID NO: 62; a LCDR1 ofSEQ ID NO:75; a LCDR2 of SEQ ID NO: 76; and a LCDR3 of SEQ ID NO: 77.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 66, a HCDR2 of SEQ ID NO: 67; a HCDR3 of SEQ ID NO: 68; a LCDR1 ofSEQ ID NO: 81; a LCDR2 of SEQ ID NO: 79; and a LCDR3 of SEQ ID NO: 77.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 86, a HCDR2 of SEQ ID NO: 87; a HCDR3 of SEQ ID NO: 88; a LCDR1 ofSEQ ID NO: 101; a LCDR2 of SEQ ID NO: 102; and a LCDR3 of SEQ ID NO:103.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 89, a HCDR2 of SEQ ID NO: 90; a HCDR3 of SEQ ID NO: 88; a LCDR1 ofSEQ ID NO: 104; a LCDR2 of SEQ ID NO: 105; and a LCDR3 of SEQ ID NO:106.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 91, a HCDR2 of SEQ ID NO: 87; a HCDR3 of SEQ ID NO: 88; a LCDR1 ofSEQ ID NO: 101; a LCDR2 of SEQ ID NO: 102; and a LCDR3 of SEQ ID NO:103.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 92, a HCDR2 of SEQ ID NO: 93; a HCDR3 of SEQ ID NO: 94; a LCDR1 ofSEQ ID NO: 107; a LCDR2 of SEQ ID NO: 105; and a LCDR3 of SEQ ID NO:103.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a heavy chainvariable region (VH) comprising the amino acid sequence of SEQ ID NO:10, and a light chain variable region (VL) comprising the amino acidsequence of SEQ ID NO: 23.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a VH comprisingthe amino acid sequence of SEQ ID NO: 36, and a VL comprising the aminoacid sequence of SEQ ID NO: 47.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a VH comprisingthe amino acid sequence of SEQ ID NO: 54, and a VL comprising the aminoacid sequence of SEQ ID NO: 23.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a VH comprisingthe amino acid sequence of SEQ ID NO: 69, and a VL comprising the aminoacid sequence of SEQ ID NO: 82.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a VH comprisingthe amino acid sequence of SEQ ID NO: 95, and a VL comprising the aminoacid sequence of SEQ ID NO: 108.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 14, and a light chain comprising theamino acid sequence of SEQ ID NO: 25.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 40, and a light chain comprising theamino acid sequence of SEQ ID NO: 49.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 58, and a light chain comprising theamino acid sequence of SEQ ID NO: 25.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 73, and a light chain comprising theamino acid sequence of SEQ ID NO: 84.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 99, and a light chain comprising theamino acid sequence of SEQ ID NO: 110.

In some embodiments, the antibody fragment (e.g., Fab) that specificallybinds to human cKIT comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 118, and a light chain comprising the amino acidsequence of SEQ ID NO: 122.

In some embodiments, the antibody fragment (e.g., Fab) that specificallybinds to human cKIT comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 118, and a light chain comprising the amino acidsequence of SEQ ID NO: 123.

In some embodiments, the antibody fragment (e.g., Fab) that specificallybinds to human cKIT comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 124, and a light chain comprising the amino acidsequence of SEQ ID NO: 128.

In some embodiments, the antibody fragment (e.g., Fab) that specificallybinds to human cKIT comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 124, and a light chain comprising the amino acidsequence of SEQ ID NO: 129.

In some embodiments, the antibody fragment (e.g., Fab) that specificallybinds to human cKIT comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 130, and a light chain comprising the amino acidsequence of SEQ ID NO: 134.

In some embodiments, the antibody fragment (e.g., Fab) that specificallybinds to human cKIT comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 130, and a light chain comprising the amino acidsequence of SEQ ID NO: 135.

In some embodiments, the antibody fragment (e.g., Fab) that specificallybinds to human cKIT comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 136, and a light chain comprising the amino acidsequence of SEQ ID NO: 140.

In some embodiments, the antibody fragment (e.g., Fab) that specificallybinds to human cKIT comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 141, and a light chain comprising the amino acidsequence of SEQ ID NO: 145.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising anamino acid sequence selected from SEQ ID NO: 119, 120 or 121, and alight chain comprising the amino acid sequence of SEQ ID NO: 25.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising anamino acid sequence selected from SEQ ID NO: 125, 126, or 127, and alight chain comprising the amino acid sequence of SEQ ID NO: 49.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising anamino acid sequence selected from SEQ ID NO: 131, 132, or 133, and alight chain comprising the amino acid sequence of SEQ ID NO: 25.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising anamino acid sequence selected from SEQ ID NO: 137, 138, or 139, and alight chain comprising the amino acid sequence of SEQ ID NO: 84.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising anamino acid sequence selected from SEQ ID NO: 142, 143, or 144, and alight chain comprising the amino acid sequence of SEQ ID NO: 110.

In some embodiments, the antibody that specifically binds to human cKITcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:12, and a light chain comprising the amino acid sequence of SEQ ID NO:25.

In some embodiments, the antibody that specifically binds to human cKITcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:38, and a light chain comprising the amino acid sequence of SEQ ID NO:49.

In some embodiments, the antibody that specifically binds to human cKITcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:56, and a light chain comprising the amino acid sequence of SEQ ID NO:25.

In some embodiments, the antibody that specifically binds to human cKITcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:71, and a light chain comprising the amino acid sequence of SEQ ID NO:84.

In some embodiments, the antibody that specifically binds to human cKITcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:97, and a light chain comprising the amino acid sequence of SEQ ID NO:110.

In some embodiments, provided herein are conjugates comprising anantibody fragment (e.g., Fab or Fab′) that specifically binds to cKIT(anti-cKIT Fab or Fab′), linked to a drug moiety (e.g., a cytotoxicagent), optionally through a linker. The anti-cKIT Fab or Fab′ can beany of the Fab or Fab′ described herein, e.g., any of the Fab or Fab′ inTable 1. As described herein, such anti-cKIT Fab′ or Fab-toxinconjugates are able to ablate human HSC cells in vitro and in vivo, butdo not cause mast cell degranulation even when crosslinked and/ormultimerized into larger complexes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line graph showing all tested anti-cKIT Fab′-(1) DAR4conjugates (see Table 2 for conjugate details) killed human stem andprogenitor cells (cKIT/CD90+ cells) in vitro with approximately equalpotency: J3 (squares); J2 (up triangles); J1 (down triangles). A controlADC, J6 (diamonds), did not kill human HSCs as compared to PBS control(circles).

FIG. 2 is a line graph showing both J4 (squares) and J5 (triangles)anti-cKIT conjugates killed mouse long term HSCs (cKIT+ cells). J5(triangles) was more potent than J4 (squares) in this mouse HSC killingassay. A control ADC, J6 (diamonds) did not kill mouse HSCs as comparedto PBS control (circles).

FIGS. 3A-3L are line graphs showing representative results of in vitrohuman mast cell degranulation assays, which used human peripheral bloodHSC-derived mast cells and beta-hexosaminidase release as the readout(assessed by absorbance at 405 nm with baseline subtraction based onreference absorbance at 620 nm). Data shown here were collected in theabsence of SCF. FIG. 3A is a line graph showing titration of eitheranti-cKIT Fab′-(1) DAR4 conjugates (closed symbols, solid lines) or fulllength anti-cKIT antibodies (open symbols, dashed lines) for variousanti-cKIT clones: anti-cKIT Ab4/Fab′4 (circles), anti-cKIT Ab3/Fab′3(squares), anti-cKIT Ab2/Fab′2 (up triangles), anti-cKIT Ab1/Fab′1 (downtriangles), and control anti-Her2 Ab/Fab′ (diamonds). FIG. 3B is a linegraph showing titration of anti-IgE as a positive control for mast celldegranulation. Mast cell degranulation was observed for allconcentrations of anti-IgE tested. FIGS. 3C-3J are line graphs showingmast cell degranulation level triggered by anti-cKIT Fab′-(1) DAR4conjugates (described in Table 2) or full length IgG anti-cKIT Abcontrols (described in Table 8) at various concentrations: absent (opendiamonds and dashed lines); 0.006 nM (triangles); 0.098 nM (diamonds);1.56 nM (circles); and 25 nM (squares), when the test agents werecross-linked using an antibody specific for the Fab portion on theantibody test agents (titrated on x-axis).

FIGS. 3C and 3D show no mast cell degranulation was triggered by J4conjugate at all tested concentrations (FIG. 3C), whereas full lengthanti-cKIT Ab4, when cross-linked, caused mast cell degranulation (FIG.3D). FIGS. 3E and 3F show no mast cell degranulation was triggered by J1conjugate at all tested concentrations (FIG. 3E), whereas full lengthanti-cKIT Ab1, when cross-linked, caused mast cell degranulation (FIG.3F). FIGS. 3G and 3H show no mast cell degranulation was triggered by J2conjugate at all tested concentrations (FIG. 3G), whereas full lengthanti-cKIT Ab2, when cross-linked, caused mast cell degranulation (FIG.3H). FIGS. 3I and 3J show no mast cell degranulation was triggered by J3conjugate at all tested concentrations (FIG. 3I), whereas full lengthanti-cKIT Ab3, when cross-linked, caused mast cell degranulation (FIG.3J). FIGS. 3K and 3L are line graphs showing no mast cell degranulationcaused by control conjugate J6 (FIG. 3K) or full length anti-Her2antibody (FIG. 3L) when cross-linked.

FIG. 4 is a dot plot showing relative numbers of human HSCs present inbone marrow of humanized NSG mice following treatment with variousagents. J7 conjugate depleted human HSCs (squares) relative to PBScontrol (circles), whereas the control J8 conjugate (diamonds) did notdeplete human HSCs in the bone marrow.

FIG. 5 is a dot plot showing relative numbers of human HSCs present inbone marrow of humanized NSG mice following treatment with variousagents. Anti-cKIT Fab′-(1) DAR4 conjugates depleted human HSCs relativeto PBS control (circles). The tested anti-cKIT conjugates (described inTable 2) were: J3 (squares); J2 (up triangles); J1 (down triangles). Thecontrol mice treated with J6 (diamonds) were not depleted of human HSCsin the bone marrow.

FIG. 6 is a bar graph showing relative numbers of HSCs present in bonemarrow of C57Bl/6 mice following treatment with various agents. Bar A=J4conjugate treated mice, Bar B=J5 conjugate treated mice, Bar C=PBStreated mice.

FIGS. 7A-71 are line graphs showing representative results of in vitrohuman mast cell degranulation assays, which used human peripheral bloodHSC-derived mast cells and beta-hexosaminidase release as the readout(assessed by absorbance at 405 nm with baseline subtraction based onreference absorbance at 620 nm). Data shown here were collected in theabsence of SCF. The line graphs show mast cell degranulation leveltriggered by antibodies or antibody fragments at various concentrations:0.006 nM (triangles); 0.098 nM (diamonds); 1.6 nM (circles); and 25 nM(squares), when the test agents were cross-linked using an antibodyspecific for the Fab portion on the antibody test agents (titrated onx-axis). FIGS. 7A-7C show that full length anti-cKIT Ab4(HC-E152C-S375C) (FIG. 7A) and anti-cKIT F(ab′4)₂ (HC-E152C) fragmentconjugated with compound (4) (FIG. 7B) caused mast cell degranulationwhen cross-linked, while no mast cell degranulation was triggered byFab4 (HC-E152C) fragment at all tested concentrations (FIG. 7C). FIGS.7D-7F show that full length anti-cKIT Ab3 (HC-E152C-S375C) (FIG. 7D) andF(ab′3)₂ (HC-E152C) fragment conjugated to compound (5) (FIG. 7E) causedmast cell degranulation when cross-linked, while no mast celldegranulation was triggered by Fab3 (E152C) fragment conjugated tocompound (4) at all tested concentrations (FIG. 7F). FIGS. 7G-71 areline graphs showing no mast cell degranulation caused by anti-Her2antibody (HC-E152C-S375C) (FIG. 7G), anti-Her2-F(ab′)₂ (HC-E152C)fragment conjugated to compound (4) (FIG. 7H), or anti-Her2-Fab(HC-E152C) fragment conjugated to compound (7) (FIG. 7I) whencross-linked.

FIGS. 8A-8O are line graphs showing representative results of in vitrohuman mast cell degranulation assays, which used human peripheral bloodHSC-derived mast cells and beta-hexosaminidase release as the readout(assessed by absorbance at 405 nm with baseline subtraction based onreference absorbance at 620 nm). Data shown here were collected in theabsence of SCF. The line graphs show mast cell degranulation leveltriggered by antibodies or antibody fragments at various concentrations:0.006 nM (triangles); 0.098 nM (diamonds); 1.6 nM (circles); and 25 nM(squares), when the test agents were cross-linked using an antibodyspecific for the Fab portion on the antibody test agents (titrated onx-axis). For reference, the cross-linker antibody alone is plotted oneach graph (open diamonds, dashed line). FIGS. 8A-8C show that fulllength anti-cKIT Ab4 (FIG. 8A) and anti-cKIT F(ab′4)₂ fragment (FIG. 8B)caused mast cell degranulation when cross-linked, while no mast celldegranulation was triggered by anti-cKIT Fab4 (HC-E152C) fragment at alltested concentrations (FIG. 8C). FIGS. 8D-8F show that full lengthanti-cKIT Ab1 (FIG. 8D) and anti-cKIT F(ab′1)₂ fragment (FIG. 8E) causedmast cell degranulation when cross-linked, while no mast celldegranulation was triggered by anti-cKIT Fab1 (HC-E152C) fragment at alltested concentrations (FIG. 8F). FIGS. 8G-81 show that full lengthanti-cKIT Ab2 (FIG. 8G) and anti-cKIT F(ab′2)₂ fragment (FIG. 8H) causedmast cell degranulation when cross-linked, while no mast celldegranulation was triggered by anti-cKIT Fab2 (HC-E152C) fragment at alltested concentrations (FIG. 8I). FIGS. 8J-8L show that full lengthanti-cKIT Ab3 (FIG. 8J) and anti-cKIT F(ab′3)₂ fragment (FIG. 8K) causedmast cell degranulation when cross-linked, while no mast celldegranulation was triggered by anti-cKIT Fab3 (HC-E152C) fragment at alltested concentrations (FIG. 8L). FIGS. 8M-80 are line graphs showing nomast cell degranulation caused by anti-Her2 antibody (FIG. 8M),anti-Her2-F(ab′)₂ fragment (FIG. 8N), or anti-Her2-Fab (HC-E152C)fragment (FIG. 8O) when cross-linked.

FIGS. 9A-9C represent in vitro killing assay results using human cells.Mobilized peripheral blood HSCs were cultured with growth factors andthe stated test agent for 7 days and viability was measured by flowcytometry and cell count. Anti-cKit Fab′ DAR4 test agents were preparedwith different Fabs: anti-cKIT Fab′1 (FIG. 9A), Fab′2 (FIG. 9B), orFab′3 (FIG. 9C). Payloads tested were C1 (open square), mc-MMAF (opencircle), C5 (diamond) or C2 (triangle). Data represent mean withstandard deviation and 3-parameter response curve fits for triplicatesmeasured in the same experiment.

FIGS. 10A-10D are line graphs showing timecourse of establishment ofdonor cell chimerism in blood samples taken from transplanted mice.C57BL/6J mice (n=5 for anti-cKit-treated group or n=2 for PBS-treatedgroup) were dosed with 10 mg/kg anti-cKit-Fab′5-DAR4-C1 (triangles), 20mg/kg anti-cKit-Fab′5-DAR4-C1 (circles), or PBS (squares) infused overseven days and then transplanted two days later with CD45.1+ donorcells. Two control animals were irradiated with 1100 RADS (diamonds) oneday prior to transplant. The line graphs show the percent donor cells(CD45.1+) measured in the population of all cells (FIG. 10A), myeloidcells (FIG. 10B), B-cells (FIG. 10C) or T-cells (FIG. 10D) by FACSanalysis of blood samples taken at each timepoint. Data represent meanwith standard error.

FIGS. 11A-11B are bar graphs showing donor cell chimerism in bloodsamples taken from transplanted mice. C57BL/6J mice (n=5 foranti-cKit-treated group or n=2 for PBS-treated group) were dosed with 10mg/kg anti-cKit-Fab′5-DAR4-C1 (horizontal stripes), 20 mg/kg anti-cKitFab′5-DAR4-C1 (vertical stripes), 40 mg/kg anti-cKit-Fab′5-DAR4-C1(solid), 40 mg/kg anti-cKit-Fab′5′-DAR4-mc-MMAF (checkered), or PBS(open, black border) infused over five days and then transplanted oneday later with CD45.1+ donor cells. Two control animals were irradiatedwith 1100 RADS (hatched) one day prior to transplant. The graphs showthe percent donor cells (CD45.1+) measured in the population of allcells (FIG. 11A) or myeloid cells (FIG. 11B) by FACS analysis of bloodsamples taken at day 28 (left bar for each group) or day 56 (right barfor each group) post-transplant. Data represent mean with standarderror.

FIGS. 12A-12B are line graphs showing timecourse of establishment ofdonor cell chimerism in blood samples taken from transplanted mice.C57BL/6J mice (n=5 for anti-cKit-treated group or n=2 for PBS-treatedgroup) were irradiated with 300 RADS and three days later were eithernot dosed (squares) or were dosed with 10 mg/kg anti-cKit-Fab′5-DAR4-C1(triangles) or 20 mg/kg anti-cKit-Fab′5-DAR4-C1 (circles) infused overthree days and then transplanted two days later with CD45.1+ donorcells. An additional group of 5 animals were only dosed with 10 mg/kganti-cKit-Fab′5-DAR4-C1 (open squares) infused over three days and thentransplanted two days later. Two control animals were irradiated with1100 RADS (diamonds) one day prior to transplant and two control animalswere untreated (open diamonds) prior to transplant. The line graphs showthe percent donor cells (CD45.1+) measured in the population of allcells (FIG. 12A) or myeloid cells (FIG. 12B) by FACS analysis of bloodsamples taken at each timepoint. Data represent mean with standarderror.

FIG. 13 is a dot plot showing relative numbers of human HSCs present inbone marrow of humanized NSG mice following treatment with variousagents. Anti-cKIT Fab′-DAR4 conjugates depleted human HSCs relative toPBS control (diamonds). The tested anti-cKIT conjugates (described inTable 2) were: JW (circles); JX (squares); JY (up triangles); JZ (downtriangles).

DETAILED DESCRIPTION

The present disclosure provides antibody drug conjugates, wherein anantibody or antibody fragment (e.g., Fab or Fab′) that specificallybinds to human cKIT is linked to a drug moiety (e.g., a cytotoxicagent), optionally through a linker. Those antibody drug conjugates canselectively deliver a cytotoxic agent to cells expressing cKIT, e.g.,hematopoietic stem cells, thereby selectively ablate those cells in apatient, e.g., a hematopoietic stem cell transplantation recipient.Preferably, the cKIT antibody drug conjugates have pharmacokineticproperties such that it will not be present and/or active in a patient'scirculation for an extended time, so they can be used for conditioninghematopoietic stem cell transplant recipients prior to hematopoieticstem cell transplantation. In some embodiments, provided herein areconjugates comprising an antibody fragment (e.g., Fab or Fab′) thatspecifically binds to cKIT, linked to a drug moiety (e.g., a cytotoxicagent), optionally through a linker. Surprisingly, the present inventorsfound that the full length anti-cKIT antibodies (e.g., full-lengthIgGs), F(ab′)₂ fragments, and toxin conjugates thereof cause mast celldegranulation, but the anti-cKIT Fab′ or Fab-toxin conjugates do notcause mast cell degranulation, even when crosslinked and/or multimerizedinto larger complexes as could be observed if a patient developed or hadpre-existing anti-drug antibodies recognizing Fab fragments. The presentdisclosure further provides pharmaceutical compositions comprising theantibody drug conjugates, and methods of making and using suchpharmaceutical compositions for ablating hematopoietic stem cells in apatient in need thereof, e.g., a hematopoietic stem cell transplantationrecipient.

Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

The term “alkyl” refers to a monovalent saturated hydrocarbon chainhaving the specified number of carbon atoms. For example, C₁₋₆alkylrefers to an alkyl group having from 1 to 6 carbon atoms. Alkyl groupsmay be straight or branched. Representative branched alkyl groups haveone, two, or three branches. Examples of alkyl groups include, but arenot limited to, methyl, ethyl, propyl (n-propyl and isopropyl), butyl(n-butyl, isobutyl, sec-butyl, and t-butyl), pentyl (n-pentyl,isopentyl, and neopentyl), and hexyl.

The term “antibody,” as used herein, refers to a protein, or polypeptidesequence derived from an immunoglobulin molecule that specifically bindsto an antigen. Antibodies can be polyclonal or monoclonal, multiple orsingle chain, or intact immunoglobulins, and may be derived from naturalsources or from recombinant sources. A naturally occurring “antibody” isa glycoprotein comprising at least two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs arranged from amino-terminus tocarboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (C1q) of the classicalcomplement system. An antibody can be a monoclonal antibody, humanantibody, humanized antibody, camelid antibody, or chimeric antibody.The antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA andIgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

“Complementarity-determining domains” or “complementary-determiningregions” (“CDRs”) interchangeably refer to the hypervariable regions ofVL and VH. The CDRs are the target protein-binding site of the antibodychains that harbors specificity for such target protein. There are threeCDRs (CDR1-3, numbered sequentially from the N-terminus) in each humanVL or VH, constituting about 15-20% of the variable domains. CDRs can bereferred to by their region and order. For example, “VHCDR1” or “HCDR1”both refer to the first CDR of the heavy chain variable region. The CDRsare structurally complementary to the epitope of the target protein andare thus directly responsible for the binding specificity. The remainingstretches of the VL or VH, the so-called framework regions, exhibit lessvariation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4.W.H. Freeman & Co., New York, 2000).

The precise amino acid sequence boundaries of a given CDR can bedetermined using any of a number of well-known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme),Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme)and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7,132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77(2003) (“IMGT” numbering scheme). For example, for classic formats,under Kabat, the CDR amino acid residues in the heavy chain variabledomain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102(HCDR3); and the CDR amino acid residues in the light chain variabledomain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97(LCDR3). Under Chothia the CDR amino acids in the VH are numbered 26-32(HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residuesin VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Bycombining the CDR definitions of both Kabat and Chothia, the CDRsconsist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102(HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56(LCDR2), and 89-97 (LCDR3) in human VL. Under IMGT the CDR amino acidresidues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2)and 93-102 (CDR3), and the CDR amino acid residues in the VL arenumbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3)(numbering according to “Kabat”). Under IMGT, the CDR regions of anantibody can be determined using the program IMGT/DomainGap Align.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3, and in somecases, CH4) confer important biological properties such as secretion,transplacental mobility, Fc receptor binding, complement binding, FcRnreceptor binding, half-life, pharmacokinetics and the like. Byconvention, the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminus is a variable region and at theC-terminus is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminal domains of the heavy and light chain,respectively.

The term “antibody fragment” or “antigen binding fragment”, as usedherein, refers to one or more portions of an antibody that retain theability to specifically interact with (e.g., by binding, sterichindrance, stabilizing/destabilizing, spatial distribution) an epitopeof an antigen (e.g., cKIT). Examples of antibody fragments include, butare not limited to, a Fab fragment, which is a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; a Fab′ fragment, which isa monovalent fragment consisting of the VL, VH, CL, CH1 domains, and thehinge region; a F(ab′)2 fragment, which is a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; a half antibody, which includes a single heavy chain and asingle light chain linked by a disulfide bridge; an one-arm antibody,which includes a Fab fragment linked to an Fc region; a CH2domain-deleted antibody, which includes two Fab fragments linked to theCH3 domain dimers (see Glaser, J Biol Chem. 2005; 280(50):41494-503); asingle-chain Fv (scFv); a disulfide-linked Fv (sdFv); a Fd fragmentconsisting of the VH and CH1 domains; a Fv fragment consisting of the VLand VH domains of a single arm of an antibody; a dAb fragment (Ward etal., Nature 341:544-546, 1989), which consists of a VH domain; and anisolated complementarity determining region (CDR), or otherepitope-binding fragments of an antibody. For example, a Fab fragmentcan include amino acid residues 1-222 (EU numbering) of the heavy chainof an antibody; whereas a Fab′ fragment can include amino acid residues1-236 (EU numbering) of the heavy chain of an antibody. The Fab or Fab′fragment of an antibody can be generated recombinantly or by enzymaticdigestion of a parent antibody. Recombinantly generated Fab or Fab′ maybe engineered to introduce amino acids for site-specific conjugationsuch as cysteines (Junutula, J. R.; et al., Nature biotechnology 2008,26, 925), pyrroline-carboxy-lysines (Ou, W. et al., Proc Natl Acad SciUSA 2011; 108(26):10437-42) or unnatural amino acids (for example Tian,F. et al., Proc Natl Acad Sci USA 2014, 111, 1766, Axup, J. Y. et al.,Proc Natl Acad Sci USA. 2012, 109, 16101. Similarly, mutations orpeptide tags can be added to facilitate conjugation throughphosphopantetheine transferases (Grunewald, J. et al., Bioconjugatechemistry 2015, 26, 2554), formyl glycine forming enzyme (Drake, P. M.et al., Bioconjugate chemistry 2014, 25, 1331), transglutaminase (Strop,P. et al., Chemistry & biology 2013, 20, 161), sortase (Beerli, R. R.;Hell, T.; Merkel, A. S.; Grawunder, U. PoS one 2015, 10, e0131177) orother enzymatic conjugation strategies. Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (“scFv”); see, e.g., Bird et al., Science 242:423-426, 1988; andHuston et al., Proc. Natl. Acad. Sci. 85:5879-5883, 1988). Such singlechain antibodies are also intended to be encompassed within the term“antigen binding fragment.” These antigen binding fragments are obtainedusing conventional techniques known to those of skill in the art, andthe fragments are screened for utility in the same manner as are intactantibodies.

Antibody fragments or antigen binding fragments can also be incorporatedinto single domain antibodies, maxibodies, minibodies, nanobodies,intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv(see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136,2005). Antigen binding fragments can be grafted into scaffolds based onpolypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No.6,703,199, which describes fibronectin polypeptide monobodies).

Antibody fragments or antigen binding fragments can be incorporated intosingle chain molecules comprising a pair of tandem Fv segments(VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.,Protein Eng. 8:1057-1062, 1995; and U.S. Pat. No. 5,641,870).

The term “monoclonal antibody” or “monoclonal antibody composition” asused herein refers to polypeptides, including antibodies and antigenbinding fragments that have substantially identical amino acid sequenceor are derived from the same genetic source. This term also includespreparations of antibody molecules of single molecular composition. Amonoclonal antibody composition displays a single binding specificityand affinity for a particular epitope.

The term “human antibody”, as used herein, includes antibodies havingvariable regions in which both the framework and CDR regions are derivedfrom sequences of human origin. Furthermore, if the antibody contains aconstant region, the constant region also is derived from such humansequences, e.g., human germline sequences, or mutated versions of humangermline sequences or antibody containing consensus framework sequencesderived from human framework sequences analysis, for example, asdescribed in Knappik et al., J. Mol. Biol. 296:57-86, 2000.

The human antibodies of the present disclosure can include amino acidresidues not encoded by human sequences (e.g., mutations introduced byrandom or site-specific mutagenesis in vitro or by somatic mutation invivo, or a conservative substitution to promote stability ormanufacturing).

The term “recognize” as used herein refers to an antibody or antigenbinding fragment thereof that finds and interacts (e.g., binds) with itsepitope, whether that epitope is linear or conformational. The term“epitope” refers to a site on an antigen to which an antibody or antigenbinding fragment of the disclosure specifically binds. Epitopes can beformed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents, whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in aunique spatial conformation. Methods of determining spatial conformationof epitopes include techniques in the art, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance (see, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)). A “paratope” is the part of the antibody whichrecognizes the epitope of the antigen.

The phrase “specifically binds” or “selectively binds,” when used in thecontext of describing the interaction between an antigen (e.g., aprotein) and an antibody, antibody fragment, or antibody-derived bindingagent, refers to a binding reaction that is determinative of thepresence of the antigen in a heterogeneous population of proteins andother biologics, e.g., in a biological sample, e.g., a blood, serum,plasma or tissue sample. Thus, under certain designated immunoassayconditions, the antibodies or binding agents with a particular bindingspecificity bind to a particular antigen at least two times thebackground and do not substantially bind in a significant amount toother antigens present in the sample. In one aspect, under designatedimmunoassay conditions, the antibody or binding agent with a particularbinding specificity binds to a particular antigen at least ten (10)times the background and does not substantially bind in a significantamount to other antigens present in the sample. Specific binding to anantibody or binding agent under such conditions may require the antibodyor agent to have been selected for its specificity for a particularprotein. As desired or appropriate, this selection may be achieved bysubtracting out antibodies that cross-react with molecules from otherspecies (e.g., mouse or rat) or other subtypes. Alternatively, in someaspects, antibodies or antibody fragments are selected that cross-reactwith certain desired molecules.

The term “affinity” as used herein refers to the strength of interactionbetween antibody and antigen at single antigenic sites. Within eachantigenic site, the variable region of the antibody “arm” interactsthrough weak non-covalent forces with antigen at numerous sites; themore interactions, the stronger the affinity.

The term “isolated antibody” refers to an antibody that is substantiallyfree of other antibodies having different antigenic specificities. Anisolated antibody that specifically binds to one antigen may, however,have cross-reactivity to other antigens. Moreover, an isolated antibodymay be substantially free of other cellular material and/or chemicals.

The term “corresponding human germline sequence” refers to the nucleicacid sequence encoding a human variable region amino acid sequence orsubsequence that shares the highest determined amino acid sequenceidentity with a reference variable region amino acid sequence orsubsequence in comparison to all other all other known variable regionamino acid sequences encoded by human germline immunoglobulin variableregion sequences. The corresponding human germline sequence can alsorefer to the human variable region amino acid sequence or subsequencewith the highest amino acid sequence identity with a reference variableregion amino acid sequence or subsequence in comparison to all otherevaluated variable region amino acid sequences. The corresponding humangermline sequence can be framework regions only, complementaritydetermining regions only, framework and complementary determiningregions, a variable segment (as defined above), or other combinations ofsequences or subsequences that comprise a variable region. Sequenceidentity can be determined using the methods described herein, forexample, aligning two sequences using BLAST, ALIGN, or another alignmentalgorithm known in the art. The corresponding human germline nucleicacid or amino acid sequence can have at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with thereference variable region nucleic acid or amino acid sequence.

A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select antibodiesspecifically immunoreactive with a protein (see, e.g., Harlow & Lane,Using Antibodies, A Laboratory Manual (1998), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity). Typically a specific or selective bindingreaction will produce a signal at least twice over the background signaland more typically at least 10 to 100 times over the background.

The term “equilibrium dissociation constant (KD [M])” refers to thedissociation rate constant (kd [s⁻¹]) divided by the association rateconstant (ka [s⁻¹, M⁻¹]). Equilibrium dissociation constants can bemeasured using any known method in the art. The antibodies of thepresent disclosure generally will have an equilibrium dissociationconstant of less than about 10⁻⁷ or 10⁻⁸ M, for example, less than about10⁻⁹ M or 10-10 M, in some aspects, less than about 10⁻¹¹ M, 10⁻¹² M or10⁻¹³ M.

The term “bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a given amount of drug administered to apatient. Bioavailability is an absolute term that indicates measurementof both the time (rate) and total amount (extent) of drug that reachesthe general circulation from an administered dosage form.

As used herein, the phrase “consisting essentially of” refers to thegenera or species of active pharmaceutical agents included in a methodor composition, as well as any excipients inactive for the intendedpurpose of the methods or compositions. In some aspects, the phrase“consisting essentially of” expressly excludes the inclusion of one ormore additional active agents other than an antibody drug conjugate ofthe present disclosure. In some aspects, the phrase “consistingessentially of” expressly excludes the inclusion of one or moreadditional active agents other than an antibody drug conjugate of thepresent disclosure and a second co-administered agent.

The term “amino acid” refers to naturally occurring, synthetic, andunnatural amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refer to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an α-carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The term “conservatively modified variant” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

For polypeptide sequences, “conservatively modified variants” includeindividual substitutions, deletions or additions to a polypeptidesequence which result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles. Thefollowing eight groups contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Glycine (G); 2) Asparticacid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see,e.g., Creighton, Proteins (1984)). In some aspects, the term“conservative sequence modifications” are used to refer to amino acidmodifications that do not significantly affect or alter the bindingcharacteristics of the antibody containing the amino acid sequence.

The term “optimized” as used herein refers to a nucleotide sequence thathas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a yeast cell, a Pichia cell, a fungal cell, aTrichoderma cell, a Chinese Hamster Ovary cell (CHO) or a human cell.The optimized nucleotide sequence is engineered to retain completely oras much as possible the amino acid sequence originally encoded by thestarting nucleotide sequence, which is also known as the “parental”sequence.

The terms “percent identical” or “percent identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refers to the extentto which two or more sequences or subsequences that are the same. Twosequences are “identical” if they have the same sequence of amino acidsor nucleotides over the region being compared. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identityover a specified region, or, when not specified, over the entiresequence), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. Optionally, the identity exists over a region that isat least about 30 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson and Lipman, Proc.Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Brent et al., Current Protocols in Molecular Biology, 2003).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410,1990, respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word lengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller, Comput. Appl.Biosci. 4:11-17, (1988) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch, J. Mol. Biol. 48:444-453, (1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

Other than percentage of sequence identity noted above, anotherindication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The term “nucleic acid” is used herein interchangeably with the term“polynucleotide” and refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, as detailed below,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,(1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem.260:2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8:91-98).

The term “operably linked” in the context of nucleic acids refers to afunctional relationship between two or more polynucleotide (e.g., DNA)segments. Typically, it refers to the functional relationship of atranscriptional regulatory sequence to a transcribed sequence. Forexample, a promoter or enhancer sequence is operably linked to a codingsequence if it stimulates or modulates the transcription of the codingsequence in an appropriate host cell or other expression system.Generally, promoter transcriptional regulatory sequences that areoperably linked to a transcribed sequence are physically contiguous tothe transcribed sequence, i.e., they are cis-acting. However, sometranscriptional regulatory sequences, such as enhancers, need not bephysically contiguous or located in close proximity to the codingsequences whose transcription they enhance.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Unless otherwise indicated, a particularpolypeptide sequence also implicitly encompasses conservatively modifiedvariants thereof.

The term “conjugate” or “antibody drug conjugate” as used herein refersto the linkage of an antibody or an antigen binding fragment thereofwith another agent, such as a chemotherapeutic agent, a toxin, animmunotherapeutic agent, an imaging probe, and the like. The linkage canbe covalent bonds, or non-covalent interactions such as throughelectrostatic forces. Various linkers, known in the art, can be employedin order to form the conjugate. Additionally, the conjugate can beprovided in the form of a fusion protein that may be expressed from apolynucleotide encoding the conjugate. As used herein, “fusion protein”refers to proteins created through the joining of two or more genes orgene fragments which originally coded for separate proteins (includingpeptides and polypeptides). Translation of the fusion gene results in asingle protein with functional properties derived from each of theoriginal proteins.

The term “subject” includes human and non-human animals. Non-humananimals include all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, dog, cow, chickens, amphibians, and reptiles.Except when noted, the terms “patient” or “subject” are used hereininterchangeably.

The term “toxin”, “cytotoxin” or “cytotoxic agent” as used herein,refers to any agent that is detrimental to the growth and proliferationof cells and may act to reduce, inhibit, or destroy a cell ormalignancy.

The term “anti-cancer agent” as used herein refers to any agent that canbe used to treat a cell proliferative disorder such as cancer, includingbut not limited to, cytotoxic agents, chemotherapeutic agents,radiotherapy and radiotherapeutic agents, targeted anti-cancer agents,and immunotherapeutic agents.

The term “drug moiety” or “payload” as used herein refers to a chemicalmoiety that is conjugated to an antibody or antigen binding fragment,and can include any therapeutic or diagnostic agent, for example, ananti-cancer, anti-inflammatory, anti-infective (e.g., anti-fungal,antibacterial, anti-parasitic, anti-viral), or an anesthetic agent. Incertain aspects, a drug moiety is selected from an Eg5 inhibitor, aV-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTorinhibitor, a microtubule stabilizer, a microtubule destabilizer, anauristatin, a dolastatin, a maytansinoid, a MetAP (methionineaminopeptidase), an inhibitor of nuclear export of proteins CRM1, aDPPIV inhibitor, an inhibitor of phosphoryl transfer reactions inmitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesininhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylatingagent, a DNA intercalator, a DNA minor groove binder, an RNA polymeraseinhibitor, an amanitin, a spliceosome inhibitor, a topoisomeraseinhibitor and a DHFR inhibitor. Methods for attaching each of these to alinker compatible with the antibodies and method of the presentdisclosure are known in the art. See, e.g., Singh et al., (2009)Therapeutic Antibodies: Methods and Protocols, vol. 525, 445-457. Inaddition, a payload can be a biophysical probe, a fluorophore, a spinlabel, an infrared probe, an affinity probe, a chelator, a spectroscopicprobe, a radioactive probe, a lipid molecule, a polyethylene glycol, apolymer, a spin label, DNA, RNA, a protein, a peptide, a surface, anantibody, an antibody fragment, a nanoparticle, a quantum dot, aliposome, a PLGA particle, a saccharide or a polysaccharide.

The term “cancer” includes primary malignant tumors (e.g., those whosecells have not migrated to sites in the subject's body other than thesite of the original tumor) and secondary malignant tumors (e.g., thosearising from metastasis, the migration of tumor cells to secondary sitesthat are different from the site of the original tumor).

The term “cKIT” (also known as KIT, PBT, SCFR, C-Kit, CD117) refers to atyrosine kinase receptor that is a member of the receptor tyrosinekinase III family. The nucleic acid and amino acid sequences of humancKIT isoforms are known, and have been published in GenBank with thefollowing Accession Nos:

-   -   NM_000222.2→NP_000213.1 Mast/stem cell growth factor receptor        Kit isoform 1 precursor;    -   NM_001093772.1→NP_001087241.1 Mast/stem cell growth factor        receptor Kit isoform 2 precursor.        Structurally, cKIT receptor is a type I transmembrane protein        and contains a signal peptide, 5 Ig-like C2 domains in the        extracellular domain and has a protein kinase domain in its        intracellular domain. As used herein, the term “cKIT” is used to        refer collectively to all naturally occurring isoforms of cKIT        protein, or a variant thereof.

The term “variant” refers to a polypeptide that has a substantiallyidentical amino acid sequence to a reference polypeptide, or is encodedby a substantially identical nucleotide sequence, and is capable ofhaving one or more activities of the reference polypeptide. For example,a variant can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or higher sequence identity to a reference polypeptide, whileretain one or more activities of the reference polypeptide.

As used herein, the terms “treat”, “treating,” or “treatment” of anydisease or disorder refer in one aspect, to ameliorating the disease ordisorder (i.e., slowing or arresting or reducing the development of thedisease or at least one of the clinical symptoms thereof). In anotheraspect, “treat”, “treating,” or “treatment” refers to alleviating orameliorating at least one physical parameter including those which maynot be discernible by the patient. In yet another aspect, “treat”,“treating,” or “treatment” refers to modulating the disease or disorder,either physically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In yet another aspect, “treat”, “treating,” or “treatment” refers topreventing or delaying the onset or development or progression of thedisease or disorder.

The term “therapeutically acceptable amount” or “therapeuticallyeffective dose” interchangeably refers to an amount sufficient to effectthe desired result (i.e., a reduction in tumor size, inhibition of tumorgrowth, prevention of metastasis, inhibition or prevention of viral,bacterial, fungal or parasitic infection). In some aspects, atherapeutically acceptable amount does not induce or cause undesirableside effects. A therapeutically acceptable amount can be determined byfirst administering a low dose, and then incrementally increasing thatdose until the desired effect is achieved. A “therapeutically effectivedosage,” of the molecules of the present disclosure can prevent theonset of, or result in a decrease in severity of, respectively, diseasesymptoms, including symptoms associated with cancer.

The term “co-administer” refers to the simultaneous presence of twoactive agents in the blood of an individual. Active agents that areco-administered can be concurrently or sequentially delivered.

The term ‘thiol-maleimide’ as used herein refers to a group formed byreaction of a thiol with maleimide, having this general formula:

where Y and Z are groups to be connected via the thiol-maleimide linkageand can comprise linker components, antibodies or payloads. Thethiol-maleimide may form the following ring opened structures

“Cleavable” as used herein refers to a linking group or linker componentthat connects two moieties by covalent connections, but breaks down tosever the covalent connection between the moieties under physiologicallyrelevant conditions, typically a cleavable linking group is severed invivo more rapidly in an intracellular environment than when outside acell, causing release of the payload to preferentially occur inside atargeted cell. Cleavage may be enzymatic or non-enzymatic, but generallyreleases a payload from an antibody without degrading the antibody.Cleavage may leave some portion of a linking group or linker componentattached to the payload, or it may release the payload without anyresidue of the linking group.

“Non-cleavable” as used herein refers to a linking group or linkercomponent that is not especially susceptible to breaking down underphysiological conditions, e.g., it is at least as stable as the antibodyor antigen binding fragment portion of the conjugate. Such linkinggroups are sometimes referred to as ‘stable’, meaning they aresufficiently resistant to degradation to keep the payload connected toantibody or antigen binding fragment until the antibody or antigenbinding fragment is itself at least partially degraded, i.e., thedegradation of the antibody or antigen binding fragment precedescleavage of the linking group in vivo. Degradation of the antibodyportion of an ADC having a stable or non-cleavable linking group mayleave some or all of the linking group, e.g., one or more amino acidgroups from an antibody, attached to the payload or drug moiety that isdelivered in vivo.

Linker-Drug Moiety (L_(B)-(D))

In one aspect, the Linker-Drug moiety of the invention comprises one ormore cytotoxins covalently attached to a linker (L_(B)), wherein the oneor more cytotoxins are independently selected from an auristatin, anamanitin, a maytansinoid and a saporin.

In another aspect, the Linker-Drug moiety of the invention comprises oneor more cytotoxins covalently attached to a linker (L_(B)), wherein theone or more cytotoxins are independently selected from an auristatin andan amanitin.

In one aspect, the Linker-Drug moiety of the invention comprises one ormore cytotoxins covalently attached to a linker (L_(B)), wherein thelinker (L_(B)) is a cleavable linker and the one or more cytotoxins areindependently selected from an auristatin, an amanitin, a maytansinoidand a saporin.

In another aspect, the Linker-Drug moiety of the invention comprises oneor more cytotoxins covalently attached to a linker (L_(B)), wherein thelinker (L_(B)) is a cleavable linker and the one or more cytotoxins areindependently selected from an auristatin and an amanitin.

In one aspect, the Linker-Drug moiety of the invention comprises one ormore cytotoxins covalently attached to a linker (L_(B)), wherein thelinker (L_(B)) is a non-cleavable linker and the one or more cytotoxinsare independently selected from an auristatin, an amanitin, amaytansinoid and a saporin.

In another aspect, the Drug moiety (D) is a protein toxin selected fromsaporin, pokeweed antiviral protein (PAP), bryodin 1, bouganin, gelonin,ricin, abrin, mistletoe lectin, modeccin, volkensin, asparin, momordin,ebulin, viscumin, Shiga toxin, diphtheria toxin (DT), or Pseudomonasexotoxin (PE). Such protein toxins are capable of killing cells byinactivating ribosome or inhibiting protein synthesis by interferingwith elongation factor 2 (EF2) function (see Kreitman et al.,Immunotoxins for targeted cancer therapy, The AAPS Journal 2006; 8 (3)Article 63; Gadadhar and Karande, Targeted Cancer Therapy: History andDevelopment of Immunotoxins, Chapter 1 of Resistance to Immunotoxins inCancer Therapy, pp 1-31). In some embodiments, the protein toxin issaporin. Such protein toxin can be covalently attached to a cleavable ornoncleavable linker (L_(B)).

In another aspect, the Linker-Drug moiety of the invention comprises oneor more cytotoxins covalently attached to a linker (L_(B)), wherein thelinker (L_(B)) is a non-cleavable linker and the one or more cytotoxinsare independently selected from an auristatin or an amanitin.

In one aspect the Linker-Drug moiety of the invention is a compoundhaving the structure of Formula (A), or stereoisomers orpharmaceutically acceptable salts thereof,

-   -   wherein:    -   R¹ is

-   -   and R³ is —OH;    -   R¹ is

-   -   and R³ is -L₅R¹⁴;    -   R² is C₁-C₆alkyl;    -   R⁴ is -L₁R¹⁴, -L₂R²⁴, -L₂R³⁴ or -L₃R⁴⁴;    -   L₁ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)—, —(CH₂)_(m)X₁(CH₂)_(m)—,        —(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —(CH₂)_(m)NHC(O)(CH₂)_(m)C(═O)NH(CH₂)_(m)—,        —((CH₂)_(m)O)_(p)(CH₂)_(m)NHC(═O)(CH₂)_(m),        —((CH₂)_(m)O)_(p)CH₂)_(m)C(═O) NH(CH₂)_(m)—,        —X₃X₄C(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—, —X₃X₄C(═O)(CH₂)_(m)—,        —X₃C(═O)(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)C(R₇)₂—, —(CH₂)_(m)C(R₇)₂SS(CH₂)_(m)NHC(═O)(CH₂)_(m)—        or —(CH₂)_(m)X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—;    -   L₂ is —((CH₂)_(m)O)_(p)(CH₂)_(m)—, —(CH₂)_(m)X₁(CH₂)_(m)—,        —(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —(CH₂)_(m)NHC(O)(CH₂)_(m)C(═O)NH(CH₂)_(m)—,        —((CH₂)_(m)O)_(p)(CH₂)_(m)NHC(═O)(CH₂)_(m),        —((CH₂)_(m)O)_(p)CH₂)_(m)C(═O) NH(CH₂)_(m)—,        —X₃X₄C(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—, —X₃X₄C(═O)(CH₂)_(m)—,        —X₃C(═O)(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)C(R₇)₂—, —(CH₂)_(m)C(R₇)₂SS(CH₂)_(m)NHC(═O)(CH₂)_(m)—        or —(CH₂)_(m)X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—;    -   L₃ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)—, —(CH₂)_(m)X₁(CH₂)_(m)—,        —(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —(CH₂)_(m)NHC(O)(CH₂)_(m)C(═O)NH(CH₂)_(m)—,        —((CH₂)_(m)O)_(p)(CH₂)_(m)NHC(═O)(CH₂)_(m),        —((CH₂)_(m)O)_(p)CH₂)_(m)C(═O) NH(CH₂)_(m)—,        —X₃X₄C(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—, —X₃X₄C(═O)(CH₂)_(m)—,        —X₃C(═O)(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)C(R₇)₂—, —(CH₂)_(m)C(R₇)₂SS(CH₂)_(m)NHC(═O)(CH₂)_(m)—        or —(CH₂)_(m)X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—;    -   L₄ is —(CH₂)_(m);    -   L₅ is —NHS(═O)₂(CH₂)_(m)X₁L₄, —NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-,        —NH((CH₂)_(m)O)_(p)(CH₂)_(m)—, —NH(CH₂)_(m)—,        —NH(CH₂)_(m)X₁(CH₂)_(m)—, —NH(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —NH(CH₂)_(m)NHC(═O)(CH₂)_(m)C(═O)NH(CH₂)_(m)—,        —NH((CH₂)_(m)O)_(p)(CH₂)_(m)NHC(═O)(CH₂)_(m),        —NH((CH₂)_(m)O)_(p)CH₂)_(m)C(═O)NH(CH₂)_(m)—,        —NH(CH₂)_(n)C(R₇)₂—,        —NH(CH₂)_(m)C(R₇)₂SS(CH₂)_(m)NHC(═O)(CH₂)_(m)— or        —NH(CH₂)_(m)X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)O)(CH₂)_(m)O)_(p)(H₂)_(m)—;

X₁ is

where the * indicates attachment point to L₄;

X₂ is

where the * indicates attachment point to L₄;

-   -   X₃ is

-   -   X₄ is

-   -   R¹⁴ is

—N₃, —ONH₂, —NR⁷C(═O)CH═CH₂, SH, —SSR¹³, —S(═O)₂(CH═CH₂),—NR⁷S(═O)₂(CH═CH₂), —NR⁷C(═O)CH₂Br, —NR⁷C(═O)CH₂I, —NHC(═O)CH₂Br,—NHC(═O)CH₂I, —C(═O)NHNH₂,

—CO₂H, —NH₂,

-   -   R²⁴ is

-   -   R³⁴ is, —N₃, —ONH₂, —NR⁷C(═O)CH═CH₂, —C(═O)NHNH₂, —CO₂H, —NH₂,

-   -   R⁴⁴ is

or —NR⁷C(═O)CH₂R⁸;

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   R⁸ is —S(CH₂)_(n)CHR⁹NH₂;    -   R⁹ is —C(═O)OR⁷;    -   each R¹⁰ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   R¹³ is 2-pyridyl or 4-pyridyl;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

In another aspect the Linker-Drug moiety of the invention is a compoundhaving the structure of Formula (B), or stereoisomers orpharmaceutically acceptable salts thereof,

wherein:

-   -   R⁵⁴ is -L₆R¹⁴, -L₇R²⁴, -L₇R³⁴ or -L₈R⁴⁴;    -   X is S(═O), S(═O)₂ or S;    -   R⁵ is H, —CH₃ or -CD₃;    -   R⁶ is —NH₂ or —OH;    -   L₆ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-,        -L₄NHC(═O)NH((CH₂)_(m)O)_(p)(CH₂)_(m)X, L₄-, -L₄NHC(═O)NH        ((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)—, —(CH₂)_(m)X₁(CH₂)_(m)—,        —(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —(CH₂)_(m)NHC(O)(CH₂)_(m)C(═O)NH(CH₂)_(m)—,        —((CH₂)_(m)O)_(p)(CH₂)_(m)NHC(═O)(CH₂)_(m),        —((CH₂)_(m)O)_(p)CH₂)_(m)C(O)NH(CH₂)_(m)—, —(CH₂)_(m)C(R₇)₂— or        —(CH₂)_(m)C(R₇)₂SS(CH₂)_(m)NHC(═O)(CH₂)_(m)—;    -   L₇ is —((CH₂)_(m)O)_(p)(CH₂)_(m)—, —(CH₂)_(m)X₁(CH₂)_(m)—,        -L₄NHC(═O)NH((CH₂)_(m)O)_(p)(CH₂)_(m)—, -L₄NHC(═O)NH        ((CH₂)_(m)O)_(p)(CH₂)_(m)—, —(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —(CH₂)_(m)NHC(O)(CH₂)_(m)C(═O)NH(CH₂)_(m)—,        —((CH₂)_(m)O)_(p)(CH₂)_(m)NHC(═O)(CH₂)_(m),        —((CH₂)_(m)O)_(p)CH₂)_(m)C(O)NH(CH₂)_(m)—, —(CH₂)_(m)C(R₇)₂—, or        —(CH₂)_(m)C(R₇)₂SS(CH₂)_(m)NHC(═O)(CH₂)_(m)—;    -   L₈ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-,        -L₄NHC(═O)NH((CH₂)_(m)O)_(p)(CH₂)_(m)X, L₄-, -L₄NHC(═O)NH        ((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)—, —(CH₂)_(m)X₁(CH₂)_(m)—,        —(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —(CH₂)_(m)NHC(O)(CH₂)_(m)C(═O)NH(CH₂)_(m)—,        —((CH₂)_(m)O)_(p)(CH₂)_(m)NHC(═O)(CH₂)_(m),        —((CH₂)_(m)O)_(p)CH₂)_(m)C(O)NH(CH₂)_(m)—, —(CH₂)_(m)C(R₇)₂— or        —(CH₂)_(m)C(R₇)₂SS(CH₂)_(m)NHC(═O)(CH₂)_(m)—;    -   L₄ is —(CH₂)_(m);    -   X₁ is

where the * indicates attachment point to L₄;

-   -   X₂ is

where the * indicates attachment point to L₄;

-   -   R¹⁴ is

—N₃, —ONH₂, —NR⁷C(═O)CH═CH₂, SH, —SSR¹³, —S(═O)₂(CH═CH₂),—NR⁷S(═O)₂(CH═CH₂), —NR¹⁷C(═O)CH₂Br, —NR⁷C(═O)CH₂I, —NHC(═O)CH₂Br,—NHC(═O)CH₂I, —C(═O)NHNH₂,

—CO₂H, —NH₂,

-   -   R²⁴ is

-   -   R³⁴ is, —N₃, —ONH₂, —NR⁷C(═O)CH═CH₂, —C(═O)NHNH₂, —CO₂H, —NH₂,

-   -   R⁴⁴ is

or —NR⁷C(═O)CH₂R⁸;

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   R⁸ is —S(CH₂)_(n)CHR⁹NH₂;    -   R⁹ is —C(═O)OR⁷;    -   each R¹⁰ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   R¹³ is 2-pyridyl or 4-pyridyl;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Certain aspects and examples of the Linker-Drug moiety of the inventionare provided in the following listing of additional, enumeratedembodiments. It will be recognized that features specified in eachembodiment may be combined with other specified features to providefurther embodiments of the present invention.

Embodiment 1

The compound of Formula (A), or a pharmaceutically acceptable saltthereof, having the structure of Formula (A-1), or a pharmaceuticallyacceptable salt thereof:

wherein: R⁴ is as defined above.

Embodiment 2

The compound of Formula (A), or a pharmaceutically acceptable saltthereof, having the structure of Formula (A-2) or Formula (A-3), or apharmaceutically acceptable salt thereof:

wherein: L₅ and R¹⁴ are as defined above.

Embodiment 3

The compound of Formula (A), or a pharmaceutically acceptable saltthereof, having the structure of Formula (A-1a), or a pharmaceuticallyacceptable salt thereof:

wherein: R⁴ is as defined above.

Embodiment 4

The compound of Formula (A), or a pharmaceutically acceptable saltthereof, having the structure of Formula (A-2a) or Formula (A-3a), or apharmaceutically acceptable salt thereof:

wherein: L₅ and R¹⁴ are as defined above.

Embodiment 5

The compound of Formula (A), Formula (A-1) or Formula (A-1a), or apharmaceutically acceptable salt thereof,

wherein:

-   -   R⁴ is -L₁R¹⁴, -L₂R²⁴, -L₂R³⁴ or -L₃R⁴⁴;    -   L₁ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)—, —X₃X₄C(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —X₃X₄C(═O)(CH₂)_(m)—, —X₃C(═O)(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—;    -   L₂ is —((CH₂)_(m)O)_(p)(CH₂)_(m)—;    -   L₃ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)—, X₃X₄C(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —X₃X₄C(═O)(CH₂)_(m)—, —X₃C(═O)(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—;    -   L₄ is —(CH₂)_(m)—;    -   X₁ is

where the * indicates attachment point to L₄;

-   -   X₂ is

where the * indicates attachment point to L₄;

-   -   X₃ is

-   -   X₄ is

-   -   R¹⁴ is

—N₃, —ONH₂, —NR⁷C(═O)CH═CH₂, SH, —S(═O)₂(CH═CH₂), —NR⁷S(═O)₂(CH═CH₂),—NR⁷C(═O)CH₂Br, —NR⁷C(═O)CH₂I, —NHC(═O)CH₂Br, —NHC(═O)CH₂I, —C(O)NHNH₂,

—CO₂H, —NH₂,

-   -   R²⁴ is

-   -   R³⁴ is, —N₃, —ONH₂, —NR⁷C(═O)CH═CH₂, —C(O)NHNH₂, —CO₂H, —NH₂,

-   -   R⁴⁴ is

or —NR⁷C(═O)CH₂R⁸;

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   R⁸ is —S(CH₂)_(n)CHR⁹NH₂;    -   R⁹ is —C(═O)OR⁷;    -   each R¹⁰ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 6

The compound of Formula (A), Formula (A-2), Formula (A-3), Formula(A-2a) or Formula (A-3a), or a pharmaceutically acceptable salt thereof,wherein:

-   -   L₄ is —(CH₂)_(m)—;    -   L₅ is —NHS(═O)₂(CH₂)_(m)X₁L₄, —NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —NH((CH₂)_(m)O)_(p)(CH₂)_(m)—        or —NH(CH₂)_(m)—;    -   X₁ is

where the * indicates attachment point to L₄; X₂ is

where the * indicates attachment point to L₄;

-   -   R¹⁴ is

—N₃, —ONH₂, —NR⁷C(O)CH═CH₂, SH, —S(═O)₂(CH═CH₂), —NHC(═O)CH₂I,—C(O)NHNH₂,

—CO₂H, —NH₂,

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   each R¹⁰ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 7

The compound of Formula (A), Formula (A-1) or Formula (A-1a), or apharmaceutically acceptable salt thereof, wherein:

-   -   R⁴ is -L₁R¹⁴;    -   L₁ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)— or —(CH₂)_(m)—;    -   L₄ is —(CH₂)_(m)—;    -   L₅ is —NHS(═O)₂(CH₂)_(m)X₁L₄;    -   X₁ is

where the * indicates attachment point to L₄;

-   -   R¹⁴ is

—ONH₂,

-   -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 8

The compound of Formula (A), Formula (A-2), Formula (A-3), Formula(A-2a) or Formula (A-3a), or a pharmaceutically acceptable salt thereof,wherein:

-   -   L₄ is —(CH₂)_(m)—;    -   L₅ is —NHS(═O)₂(CH₂)_(m)X₁L₄;    -   X₁ is

where the * indicates attachment point to L₄;

-   -   R¹⁴ is

—ONH₂,

-   -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 9

The compound of Formula (A) selected from:

Embodiment 10

The compound of Formula (B), or a pharmaceutically acceptable saltthereof, having the structure of Formula (B-1), or a pharmaceuticallyacceptable salt thereof:

wherein: R⁵⁴, R⁵ and R⁶, are as defined above.

Embodiment 11

The compound of Formula (B), or a pharmaceutically acceptable saltthereof, having the structure of Formula (B-1a), or a pharmaceuticallyacceptable salt thereof:

wherein: R⁵⁴, R⁵ and R⁶, are as defined above.

Embodiment 12

The compound of Formula (B), Formula (B-1) or Formula (B-1a), or apharmaceutically acceptable salt thereof, wherein:

-   -   R⁵⁴ is -L₆R¹⁴, -L₇R²⁴, -L₇R³⁴ or -L₈R⁴⁴;    -   R⁵ is H, —CH₃ or -CD₃;    -   R⁶ is —NH₂ or —OH;    -   L₆ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-,        -L₄NHC(═O)NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-, -L₄NHC(═O)NH        ((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)— or        —(CH₂)_(m)—;    -   L₇ is —((CH₂)_(m)O)_(p)(CH₂)_(m)—;    -   L₈ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)—;    -   L₄ is —(CH₂)_(m)—;    -   X₁ is

where the * indicates attachment point to L₄;

-   -   X₂ is

where the * indicates attachment point to L₄;

-   -   R¹⁴ is

—N₃, —ONH₂, —NR⁷C(═O)CH═CH₂, SH, —S(═O)₂(CH═CH₂), —NR⁷S(═O)₂(CH═CH₂),—NR⁷C(═O)CH₂Br, —NR⁷C(═O)CH₂I, —NHC(═O)CH₂Br, —NHC(═O)CH₂I, —C(O)NHNH₂,

—CO₂H, —NH₂,

-   -   R²⁴ is

-   -   R³⁴ is, —N₃, —ONH₂, —NR⁷C(═O)CH═CH₂, —C(O)NHNH₂, —CO₂H, —NH₂,

-   -   R⁴⁴ is

or —NR⁷C(═O)CH₂R⁸;

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   R⁸ is —S(CH₂)_(n)CHR⁹NH₂;    -   R⁹ is —C(═O)OR⁷;    -   each R¹⁰ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH; each R¹¹ is independently selected from H, C₁-C₆alkyl,        F, Cl, —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 13

The compound of Formula (B), Formula (B-1) or Formula (B-1a), or apharmaceutically acceptable salt thereof, wherein:

-   -   R⁵⁴ is -L₆R¹⁴;    -   R⁵ is —CH₃;    -   R⁶ is —NH₂;    -   L₆ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)—,        -L₄NHC(═O)NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-, -L₄NHC(═O)NH        ((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, or —(CH₂)_(m)—;    -   L₄ is —(CH₂)_(m)—;    -   X₁ is

where the * indicates attachment point to L₄;

-   -   R¹⁴ is

—ONH₂,

-   -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 14

The compound of Formula (B) selected from:

In another aspect the Linker-Drug moiety of the invention is selectedfrom:

In another aspect the Linker-Drug moiety of the invention is selectedfrom:

Antibody Drug Conjugates

The present disclosure provides antibody drug conjugates, wherein anantibody or antibody fragment (e.g., Fab or Fab′) that specificallybinds to cKIT is linked to a drug moiety (e.g., a cytotoxic agent),optionally through a linker. In one aspect, the antibody or antibodyfragment (e.g., Fab or Fab′) is linked, via covalent attachment by alinker, to a drug moiety that is a cytotoxic agent.

The antibody drug conjugates can selectively deliver a cytotoxic agentto cells expressing cKIT, e.g., hematopoietic stem cells, therebyselectively ablate those cells in a patient, e.g., a hematopoietic stemcell transplantation recipient. Preferably, the cKIT antibody drugconjugates have short half-life and will be cleared from a patient'scirculation so they can be used for conditioning hematopoietic stem celltransplant recipients prior to hematopoietic stem cell transplantation.

In some embodiments, the cKIT antibody drug conjugates disclosed hereinare modified to have reduced ability to induce mast cell degranulation,even when cross-linked and/or multimerized into larger complexes. Forexample, the cKIT antibody drug conjugates disclosed herein are modifiedto have a reduced ability to induce mast cell degranulation that is, isabout, or is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99% reduced in comparison to a full-length cKIT antibody, an F(ab′)₂ oran F(ab)₂ fragment, or conjugate thereof, even when cross-linked and/ormultimerized into larger complexes. In some embodiments, the cKITantibody drug conjugates disclosed herein may comprise an anti-cKIT Fabor Fab′ fragment. In some embodiments, the anti-cKIT antibody drugconjugates disclosed herein may have minimal activity to induce mastcell degranulation, e.g., a baseline corrected O.D. readout of less than0.25, e.g., less than 0.2, less than 0.15, or less than 0.1, in abeta-hexosaminidase release assay, even when cross-linked and/ormultimerized into larger complexes.

In some embodiments, provided herein are conjugates comprising anantibody fragment (e.g., Fab or Fab′) that specifically binds to cKIT(anti-cKIT Fab or Fab′), linked to a drug moiety (e.g., a cytotoxicagent), optionally through a linker. As described herein, such anti-cKITFab′ or Fab-toxin conjugates are able to ablate human HSC cells in vitroand in vivo, but do not cause mast cell degranulation even whencrosslinked and/or multimerized into larger complexes.

In one aspect, the disclosure provides for an conjugate of Formula (I):

A-(L_(B)-(D)_(n))_(y)  Formula (I);

wherein:

A is an antibody fragment (e.g., Fab or Fab′) that specifically binds tohuman cKIT;

L_(B) is a linker;

D is a cytotoxic agent;

n is an integer from 1 to 10, and

y is an integer from 1 to 10,

where the Linker-Drug moiety (L_(B)-(D)_(n)) is covalently attached tothe antibody fragment (A).

In one aspect, the present disclosure is directed to a conjugate ofFormula (II):

A₁ is an antibody fragment (e.g., Fab or Fab′) or chain (e.g. HC or LC)that specifically binds to human cKIT;A₂ is an antibody fragment (e.g., Fab or Fab′) or chain (e.g. HC or LC)that specifically binds to human cKIT;LB is a linker;D is a cytotoxic agent, andn is an integer from 1 to 10,where the Linker-Drug moiety (L_(B)-(D)_(n)) covalently couples theantibody fragments A₁ and A₂.

In one aspect, the one of more drug moieties, D, in the conjugates ofFormula (I) and Formula (II) are independently selected from anauristatin, an amanitin, a maytansinoid and a saporin.

In another aspect, the one of more drug moieties, D, in the conjugatesof Formula (I) are independently selected from an auristatin and anamanitin.

In the conjugates of Formula (I), one or more Linker-Drug moiety(L_(B)-(D)_(n)) can be covalently attached to the antibody fragment, A(e.g. Fab or Fab′), thereby covalently attaching one or more drugmoieties, D, to the antibody fragment, A (e.g. Fab or Fab′), throughlinker, L_(B). L_(B) is any chemical moiety that is capable of linkingthe antibody fragment, A (e.g. Fab or Fab′) to one or more drugmoieties, D. The conjugates of Formula (I), wherein one or more drugmoieties, D, are covalently linked to an antibody fragment, A (e.g. Fabor Fab′), can be formed using a bifunctional or multifunctional linkerreagent having one or more reactive functional groups that are the sameor different. One of the reactive functional groups of the bifunctionalor multifunctional linker reagent is used to react with a group on theantibody fragment, A, by way of example, a thiol or an amine (e.g. acysteine, an N-terminus or amino acid side chain such as lysine) to forma covalent linkage with one end of the linker L_(B). Such reactivefunctional groups of the bifunctional or multifunctional linker reagentinclude, but are not limited to, a maleimide, a thiol and an NHS ester.The other reactive functional group or groups of the bifunctional ormultifunctional linker reagent are used to covalently attached one ormore drug moieties, D, to linker L_(B).

In the conjugates of Formula (II), a ketone bridge is formed by reactionof pendent thiols on antibody fragments A₁ and A₂ and a1,3-dihaloacetone, such as 1,3-dichloroacetone, 1,3-dibromoacetone,1,3-diiodoacetone, and bissulfonate esters of 1, 3-dihydroxyacetone,which thereby covalently couples the antibody fragments A₁ and A₂. Thisketone bridge moiety is used to covalently attach one or more drugmoieties, D, to the antibody fragments A, and A₂ through a linker L_(B).L_(B) is any chemical moiety that is capable of linking the antibodyfragment, A, and A₂ to one or more drug moieties, D. The conjugates ofFormula (II), wherein one or more drug moieties, D, are covalentlylinked to antibody fragments A, and A₂, can be formed using abifunctional or multifunctional linker reagent having one or morereactive functional groups that are the same or different. In anembodiment, one the reactive functional groups of the bifunctional ormultifunctional linker reagent is an alkoxyamine which is used to reactwith the ketone bridge to form an oxime linkage with one end of thelinker L_(B), and the other reactive functional group or groups of thebifunctional or multifunctional linker reagent are used to covalentlyattached one or more drug moieties, D, to linker L_(B). In anotherembodiment, one the reactive functional groups of the bifunctional ormultifunctional linker reagent is an hydrazine which is used to reactwith the ketone bridge to form a hydrazone linkage with one end of thelinker L_(B), and the other reactive functional group or groups of thebifunctional or multifunctional linker reagent are used to covalentlyattached one or more drug moieties, D, to linker L_(B).

In one aspect, L_(B) is a cleavable linker. In another aspect, L_(B) isa non-cleavable linker. In some aspects, L_(B) is an acid-labile linker,photo-labile linker, peptidase cleavable linker, esterase cleavablelinker, glycosidase cleavable linker, phosphodiesterase cleavablelinker, a disulfide bond reducible linker, a hydrophilic linker, or adicarboxylic acid based linker.

In another aspect, the drug moiety (D) is a protein toxin selected fromsaporin, pokeweed antiviral protein (PAP), bryodin 1, bouganin, gelonin,ricin, abrin, mistletoe lectin, modeccin, volkensin, asparin, momordin,ebulin, viscumin, Shiga toxin, diphtheria toxin (DT), or Pseudomonasexotoxin (PE). Such protein toxins are capable of killing cells byinactivating ribosome or inhibiting protein synthesis by interferingwith elongation factor 2 (EF2) function (see Kreitman et al.,Immunotoxins for targeted cancer therapy, The AAPS Journal 2006; 8 (3)Article 63; Gadadhar and Karande, Targeted Cancer Therapy: History andDevelopment of Immunotoxins, Chapter 1 of Resistance to Immunotoxins inCancer Therapy, pp 1-31). In some embodiments, the protein toxin issaporin. The protein toxin can be attached to the anti-cKIT antibodyfragment (A) covalently through a cleavable or noncleavable linker(L_(B)). In some embodiments, the protein toxin is linked to theanti-cKIT antibody fragment through a disulfide or thioether linkage.

While the drug to antibody ratio has an exact integer value for aspecific conjugate molecule (e.g., the product of n and y in Formula (I)and “n” in Formula (II)), it is understood that the value will often bean average value when used to describe a sample containing manymolecules, due to some degree of inhomogeneity, typically associatedwith the conjugation step. The average loading for a sample of aconjugate is referred to herein as the drug to antibody (or Fab′) ratio,or “DAR.” In some aspects, the DAR is between about 1 and about 5, andtypically is about 1, 2, 3, or 4. In some aspects, at least 50% of asample by weight is compound having the average DAR plus or minus 2, andpreferably at least 50% of the sample is a conjugate that contains theaverage DAR plus or minus 1. Other aspects include conjugates whereinthe DAR is about 2. In some aspects, a DAR of ‘about y’ means themeasured value for DAR is within 20% of the product of n and y inFormula (I). In some aspects, a DAR of ‘about n’ means the measuredvalue for DAR is within 20% of n in Formula (II).

In one aspect, the average molar ratio of the drug to the antibodyfragment (Fab or Fab′) in the conjugates of Formula (I) (i.e., averagevalue of the product of n and y, also known as drug to antibody ratio(DAR)) is about 1 to about 10, about 1 to about 6 (e.g., 0.9, 1.0, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0), about 1 to about 5, about 1.5 toabout 4.5, or about 2 to about 4.

In one aspect, the average molar ratio of the drug to the antibodyfragments A₁ and A₂ in the conjugates of Formula (II) (i.e., averagevalue of n, also known as drug to antibody ratio (DAR)) is about 1 toabout 10, about 1 to about 6 (e.g., 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6.0), about 1 to about 5, about 1.5 to about 4.5, or about 2to about 4.

In one aspect provided by the disclosure, the conjugate hassubstantially high purity and has one or more of the following features:(a) greater than about 90% (e.g., greater than or equal to about 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), preferably greaterthan about 95%, of conjugate species are monomeric, (b) unconjugatedlinker level in the conjugate preparation is less than about 10% (e.g.,less than or equal to about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%)(relative to total linker), (c) less than 10% of conjugate species arecrosslinked (e.g., less than or equal to about 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, 1%, or 0%), (d) free drug (e.g., auristatin, amanitin,maytansinoid or saporin) level in the conjugate preparation is less thanabout 2% (e.g., less than or equal to about 1.5%, 1.4%, 1.3%, 1.2%,1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%)(mol/mol relative to total cytotoxic agent).

In one aspect the conjugates of the invention have the structure ofFormula (C):

wherein:

-   -   A represents an antibody fragment (e.g., Fab or Fab′) that        specifically binds to human cKIT;    -   y is an integer from 1 to 10;    -   R² is C₁-C₆alkyl;    -   L₂₀ is -L₁R⁴⁰;    -   L₁ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)—, —(CH₂)_(m)X(CH₂)_(m)—, —(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —(CH₂)_(m)NHC(═O)(CH₂)_(m)C(═O)NH(CH₂)_(m)—,        —((CH₂)_(m)O)_(p)(CH₂)_(m)NHC(═O)(CH₂)_(m),        —((CH₂)_(m)O)_(p)CH₂)_(m)C(═O)NH(CH₂)_(m)—,        X₃X₄C(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—, —X₃X₄C(═O)(CH₂)_(m)—,        —X₃C(═O)(CH₂)_(n)NHC(═O)(CH₂)_(m)—,        —X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)C(R₇)₂—, —(CH₂)_(m)C(R₇)₂SS(CH₂)_(m)NHC(═O)(CH₂)_(m)—        or —(CH₂)_(m)X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—;    -   L₄ is —((CH₂)_(m);    -   X₁ is

where the * indicates attachment point to L₄;

-   -   X₂ is

where the * indicates attachment point to L₄;

-   -   X₃ is

-   -   R⁴⁰ is

—NR⁷C(═O)CH₂—, —NHC(═O)CH₂—, —S(═O)₂CH₂CH₂—, —(CH₂)₂S(═O)₂CH₂CH₂—,—NR⁷S(═O)₂CH₂CH₂, —NR⁷C(═O)CH₂CH₂—, —NH—, —C(═O)—, —NHC(═O)—,—CH₂NHCH₂CH₂—, —NHCH₂CH₂—, —S—,

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   each R¹⁰ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   each R¹⁵ is independently selected from H, —CH₃ and phenyl;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

In another aspect the conjugates of the invention have the structure ofFormula (D):

wherein:

-   -   A represents an antibody fragment (e.g., Fab or Fab′) that        specifically binds to human cKIT;    -   y is an integer from 1 to 10;    -   R¹ is

-   -   R² is C₁-C₆alkyl;    -   L₃₀ is -L₅R⁴⁰;    -   L₄ is —((CH₂)_(m);    -   L₅ is —NHS(═O)₂(CH₂)_(m)X₁L₄, —NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-,        —NH((CH₂)_(m)O)_(p)(CH₂)_(m)—, —NH(CH₂)_(m)—,        —NH(CH₂)_(m)X₁(CH₂)_(m)—, —NH(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —NH(CH₂)_(m)NHC(═O)(CH₂)_(m)C(═O)NH(CH₂)_(m)—,        —NH((CH₂)_(m)O)_(p)(CH₂)_(m)NHC(═O)(CH₂)_(m),        —NH((CH₂)_(m)O)_(p)CH₂)_(m)C(═O)NH(CH₂)_(m)—,        —NH(CH₂)_(n)C(R₇)₂—,        —NH(CH₂)_(m)C(R₇)₂SS(CH₂)_(m)NHC(═O)(CH₂)_(m)— or        —NH(CH₂)_(m)X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—;    -   X₁ is

where the * indicates attachment point to L₄;

-   -   X₂ is

where the * indicates attachment point to L₄;

-   -   X₃ is

-   -   X₄ is

-   -   R⁴⁰ is

—NR⁷C(═O)CH₂—, —NHC(═O)CH₂—, —S(═O)₂CH₂CH₂—, —(CH₂)₂S(═O)₂CH₂CH₂—,—NR⁷S(═O)₂CH₂CH₂, —NR⁷C(═O)CH₂CH₂—, —NH—, —C(═O)—, —NHC(═O)—,—CH₂NHCH₂CH₂—, —NHCH₂CH₂—, —S—,

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   each R¹⁰ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   each R¹⁵ is independently selected from H, —CH₃ and phenyl;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and each p is independently selected from 1, 2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13 and 14.

In another aspect the conjugates of the invention have the structure ofFormula (E):

wherein:

-   -   A represents an antibody fragment (e.g., Fab or Fab′) that        specifically binds to human cKIT;    -   y is an integer from 1 to 10;    -   X is S(═O), S(═O)₂ or S;    -   R⁵ is H, —CH₃ or -CD₃;    -   R⁶ is —NH₂ or —OH;    -   L₄₀ is -L₆R⁴⁰;    -   L₆ is is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-,        -L₄NHC(═O)NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-, -L₄NHC(═O)NH        ((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)—, —(CH₂)_(m)X(CH₂)_(m)—, —(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —(CH₂)_(m)NHC(═O)(CH₂)_(m)OC(═)NH(CH₂)_(m)—,        —((CH₂)O)_(p)(CH₂)_(m)NHC(═O)(CH₂)_(m),        —((CH₂)_(m)O)_(p)CH₂)_(m)C(═O)NH(CH₂)_(m)—, —(CH₂)_(m)C(R₇)₂— or        —(CH₂)_(m)C(R₇)₂SS(CH₂)_(m)NHC(═O)(CH₂)_(m)—;    -   L₄ is —((CH₂)_(m);    -   X₁ is

where the * indicates attachment point to L₄;

-   -   X₂ is

where the * indicates attachment point to L₄;

-   -   R⁴⁰ is

—NR⁷C(═O)CH₂—, —NHC(═O)CH₂—, —S(═O)₂CH₂CH₂—, —(CH₂)₂S(═O)₂CH₂CH₂—,—NR⁷S(═O)₂CH₂CH₂, —NR⁷C(═O)CH₂CH₂—, —NH—, —C(═O)—, —NHC(═O)—,—CH₂NHCH₂CH₂—, —NHCH₂CH₂—, —S—,

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   each R¹⁰ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   each R¹⁵ is independently selected from H, —CH₃ and phenyl;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Certain aspects and examples of the conjugates of the invention areprovided in the following listing of additional, enumerated embodiments.It will be recognized that features specified in each embodiment may becombined with other specified features to provide further embodiments ofthe present invention.

Embodiment 15

The conjugate having the structure of Formula (C) is a conjugate havinghas the structure of Formula (C-1):

wherein: A, y, and L₂₀ are as defined above.

Embodiment 16

The conjugate having the structure of Formula (C) or Formula (C-1)wherein:

-   -   A represents an antibody fragment (e.g., Fab or Fab′) that        specifically binds to human cKIT;    -   y is an integer from 1 to 10;    -   L₂₀ is -L₁R⁴⁰;    -   L₁ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —(CH₂)_(m)—, —X₃X₄C(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—,        —X₃X₄C(═O)(CH₂)_(m)—, —X₃C(═O)(CH₂)_(m)NHC(═O)(CH₂)_(m)—,        —X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—;    -   L₄ is —((CH₂)_(m);    -   X₁ is

where the * indicates attachment point to L₄;

-   -   X₂ is

where the * indicates attachment point to L₄;

-   -   X₃ is

-   -   X₄ is

-   -   R⁴⁰ is

—NR⁷C(═O)CH₂—, —NHC(═O)CH₂—, —S(═O)₂CH₂CH₂—, —(CH₂)₂S(═O)₂CH₂CH₂—,—NR⁷S(═O)₂CH₂CH₂, —NR⁷C(═O)CH₂CH₂—, —NH—, —C(═O)—, —NHC(═O)—,—CH₂NHCH₂CH₂—, —NHCH₂CH₂—, —S—,

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   each R¹⁰ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 17

The conjugate having the structure of Formula (C) or Formula (C-1),wherein:

-   -   A represents an antibody fragment (e.g., Fab or Fab′) that        specifically binds to human cKIT;    -   y is an integer from 1 to 10;    -   L₂₀ is -L₁R⁴⁰;    -   L₁ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)— or        —(CH₂)_(m)—;    -   L₄ is —((CH₂)_(m);    -   X₁ is

where the * indicates attachment point to L₄;

-   -   X₂ is

where the * indicates attachment point to L₄;

-   -   R⁴⁰ is

—NR⁷C(═O)CH—, NHC(═O)CH₂—, —S(═O)₂CH₂CH₂—, —(CH₂)₂S(═O)₂CH₂CH₂—,—NR⁷S(═O)₂CH₂CH₂, —NR⁷C(═O)CH₂CH₂—, —NH—, —C(═O)—, —NHC(═O)—,—CH₂NHCH₂CH₂—, —NHCH₂CH₂—, —S—,

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   each R¹⁰ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 18

The conjugate having the structure of Formula (C) or Formula (C-1),wherein:

-   -   A represents an antibody fragment (e.g., Fab or Fab′) that        specifically binds to human cKIT;    -   y is an integer from 1 to 10;    -   L₂₀ is -L₁R⁴⁰;    -   L₁ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)— or —(CH₂)_(m)—;    -   L₄ is —((CH₂)_(m);    -   X₁ is

where the * indicates attachment point to L₄;

-   -   R⁴⁰ is

-   -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 19

The conjugate having the structure of Formula (C) or Formula (C-1)selected from:

Embodiment 20

The conjugate having the structure of Formula (D) is a conjugate havinghas the structure of Formula (D-1) or Formula (D-2):

wherein: A, y and L₃₀ are as defined above.

Embodiment 21

The conjugate having the structure of Formula (D) is a conjugate havinghas the structure of Formula (D-1a) or Formula (D-2a):

wherein: A, y and L₃₀ are as defined above.

Embodiment 22

The conjugate having the structure of Formula (D), Formula (D-1),Formula (D-2), Formula (D-1a) or Formula (D-2a), wherein:

-   -   A represents an antibody fragment (e.g., Fab or Fab′) that        specifically binds to human cKIT;    -   y is an integer from 1 to 10;    -   L₃₀ is -L₅R⁴⁰;    -   L₄ is —((CH₂)_(m);    -   L₅ is —NHS(═O)₂(CH₂)_(m)X₁L₄, —NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —NH((CH₂)_(m)O)_(p)(CH₂)_(m)—        or —NH(CH₂)_(m)—;    -   X₁ is

where the * indicates attachment point to L₄;

-   -   X₂ is

where the * indicates attachment point to L₄;

-   -   R⁴⁰ is

—NR⁷C(═O)CH₂—, —NHC(═O)CH₂—, —S(═O)₂CH₂CH₂—, —(CH₂)₂S(═O)₂CH₂CH₂—,—NR⁷S(═O)₂CH₂CH₂, —NR⁷C(═O)CH₂CH₂—, —NH—, —C(═O)—, —NHC(═O)—,—CH₂NHCH₂CH₂—, —NHCH₂CH₂—, —S—,

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   each R¹⁰ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 23

The conjugate having the structure of Formula (D), Formula (D-1),Formula (D-2), Formula (D-1a) or Formula (D-2a):

wherein:

-   -   A represents an antibody fragment (e.g., Fab or Fab′) that        specifically binds to human cKIT;    -   y is an integer from 1 to 10;    -   L₃₀ is -L₅R⁴⁰;    -   L₄ is —((CH₂)_(m);    -   L₅ is —NHS(═O)₂(CH₂)_(m)X₁L₄, —NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —NH((CH₂)_(m)O)_(p)(CH₂)_(m)—        or —NH(CH₂)_(m)—;    -   X₁ is

where the * indicates attachment point to L₄;

-   -   X₂ is

where the * indicates attachment point to L₄;

-   -   R⁴⁰ is

—NR⁷C(═O)CH₂—, —NHC(═O)CH₂—, —S(═O)₂CH₂CH₂—, —(CH₂)₂S(═O)₂CH₂CH₂—,—NR⁷S(═O)₂CH₂CH₂, —NR⁷C(═O)CH₂CH₂—, —NH—, —C(═O)—, —NHC(═O)—,—CH₂NHCH₂CH₂—, —NHCH₂CH₂—, —S—,

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   each R¹⁰ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 24

The conjugate having the structure of Formula (D), Formula (D-1),Formula (D-2), Formula (D-1a) or Formula (D-2a), wherein:

-   -   A represents an antibody fragment (e.g., Fab or Fab′) that        specifically binds to human cKIT;    -   y is an integer from 1 to 10;    -   L₃₀ is -L₅R⁴⁰;    -   L₄ is —((CH₂)_(m);    -   L₅ is —NHS(═O)₂(CH₂)_(m)X₁L₄;    -   X₁ is

where the * indicates attachment point to L₄;

-   -   R⁴⁰ is

-   -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 25

The conjugate having the structure of Formula (D), Formula (D-1),Formula (D-2), Formula (D-1a) or Formula (D-2a) selected from:

Embodiment 26

The conjugate having the structure of Formula (E) is a conjugate havinghas the structure of Formula (E-1):

wherein: A, y, R⁵, R⁶ and L₄₀ are as defined above.

Embodiment 27

The conjugate having the structure of Formula (E) is a conjugate havinghas the structure of Formula (E-1a):

wherein: A, y, R⁵, R⁶ and L₄₀ are as defined above.

Embodiment 28

The conjugate having the structure of Formula (E), Formula (E-1) orFormula (E-1a), wherein:

-   -   A represents an antibody fragment (e.g., Fab or Fab′) that        specifically binds to human cKIT;    -   y is an integer from 1 to 10;    -   R⁵ is H, —CH₃ or -CD₃;    -   R⁶ is —NH₂ or —OH;    -   L₄₀ is -L₆R⁴⁰;    -   L₆ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-,        -L₄NHC(═O)NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-, -L₄NHC(═O)NH        ((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)— or        —(CH₂)_(m)—;    -   L₄ is —((CH₂)_(m);    -   X₁ is

where the * indicates attachment point to L₄;

-   -   X₂ is

where the * indicates attachment point to L₄;

-   -   R⁴⁰ is

—NR⁷C(═O)CH₂—, —NHC(═O)CH₂—, —S(═O)₂CH₂CH₂—, —(CH₂)₂S(═O)₂CH₂CH₂—,—NR⁷S(═O)₂CH₂CH₂, —NR⁷C(═O)CH₂CH₂—, —NH—, —C(═O)—, —NHC(═O)—,—CH₂NHCH₂CH₂—, —NHCH₂CH₂—, —S—,

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   each R¹⁰ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 29

The conjugate having the structure of Formula (E), Formula (E-1) orFormula (E-1a), wherein:

-   -   A represents an antibody fragment (e.g., Fab or Fab′) that        specifically binds to human cKIT;    -   y is an integer from 1 to 10;    -   R⁵ is H, —CH₃ or -CD₃;    -   R⁶ is —NH₂ or —OH;    -   L₄₀ is -L₆R⁴⁰;    -   L₆ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-,        -L₄NHC(═O)NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-, -L₄NHC(═O)NH        ((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)— or        —(CH₂)_(m)—;    -   L₄ is —((CH₂)_(m);    -   X₁ is

where the * indicates attachment point to L₄;

-   -   X₂ is

where the * indicates attachment point to L₄;

-   -   R⁴⁰ is

—NR⁷C(═O)CH₂—, —NHC(═O)CH₂—, —S(═O)₂CH₂CH₂—, —(CH₂)₂S(═O)₂CH₂CH₂—,—NR⁷S(═O)₂CH₂CH₂, —NR⁷C(═O)CH₂CH₂—, —NH—, —C(═O)—, —NHC(═O)—,—CH₂NHCH₂CH₂—, —NHCH₂CH₂—, —S—,

-   -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        and —OH;    -   each R¹¹ is independently selected from H, C₁-C₆alkyl, F, Cl,        —NH₂, —OCH₃, —OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH;    -   each R¹² is independently selected from H, C₁₋₆alkyl, fluoro,        benzyloxy substituted with —C(═O)OH, benzyl substituted with        —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH and C₁₋₄alkyl        substituted with —C(═O)OH;    -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 30

The conjugate having the structure of Formula (E), Formula (E-1) orFormula (E-1a), wherein:

-   -   A represents an antibody fragment (e.g., Fab or Fab′) that        specifically binds to human cKIT;    -   y is an integer from 1 to 10;    -   R⁵ is —CH₃;    -   R⁶ is —NH₂;    -   L₄₀ is -L₆R⁴⁰;    -   L₆ is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,        —((CH₂)_(m)O)_(p)(CH₂)_(m)—,        -L₄NHC(═O)NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-, -L₄NHC(═O)NH        ((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, or —(CH₂)_(m)—;    -   L₄ is —((CH₂)_(m);    -   X₁ is

where the * indicates attachment point to L₄;

-   -   R⁴⁰ is

-   -   each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9        and 10, and    -   each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13 and 14.

Embodiment 31

The conjugate having the structure of Formula (E), Formula (E-1) orFormula (E-1a) selected from:

In another aspect of the antibody drug conjugate of the invention isselected from:

-   -   wherein A represents an antibody fragment (e.g., Fab or Fab′)        that specifically binds to human cKIT,    -   and y is an integer from 1 to 10.

In another aspect of the antibody drug conjugate of the invention isselected from:

-   -   wherein A represents an antibody fragment (e.g., Fab or Fab′)        that specifically binds to human cKIT,    -   and y is an integer from 1 to 10.

Synthesis of Exemplary Linker-Drug Compounds Example 1: Synthesis of(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((1R,3S,4S)-2-(3-(2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoyl)-2-azabicyclo[2.2.1]heptane-3-carboxamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid (C1)

Step 1:

To a solution of BocVal-Dil-Dap-OH (1.00 g, 1.75 mmol) inN,N-dimethylformamide (DMF, 20.0 mL) at 0° C. were added N,N-diisopropylethylamine (DIEA, 0.677 g, 5.25 mmol) and1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU)(0.731 g, 1.93 mmol). The resultingsolution was then stirred for 5 minutes and added to a solution ofL-phenylalanine methyl ester HCl salt (0.377 g, 1.75 mmol) and DIEA(0.226 g, 1.75 mmol) in DMF (5.0 mL) at 0° C. The reaction mixture waswarmed to room temperature, stirred for an additional 30 minutes andthen concentrated. The residue was purified by reverse phase HPLC usingthe ISCO system, C18 column, eluted with 20-90% acetonitrile-water toobtain BocVal-Dil-Dap-PheOMe: MS m/z 733.4 (M+1); retention time 1.47minutes.

Step 2:

To a solution of BocVal-Dil-Dap-PheOMe (0.683 g, 0.932 mmol) obtained instep 1 in methanol (20 mL) was added HCl (4N in 1, 4-dioxane, 16 mL).The reaction mixture was stirred at room temperature for 7 hours andconcentrated. The residue was dissolved in dioxane and lyophilized toobtain Val-Dil-Dap-PheOMe HCl salt: MS m/z 633.4 (M+1); retention time0.96 minutes.

Step 3:

(1R,3S,4S)—N-Boc-2-azabicyclo[2.2.1]heptane-3-carboxylic acid (12.6 mg,0.052 mmol) was dissolved in DMF (1 mL) in a 15 ml round bottom flask.DIEA (12.3 mg, 0.095 mmol) and HATU (19 mg, 0.050 mmol) were added. Thereaction mixture was stirred for 10 minutes and Val-Dil-Dap-PheOMe HClsalt (30 mg, 0.090 mmol) in DMF (1.0 mL) was added. The reaction mixturewas stirred for 1 hour. LCMS analysis indicated the reaction wascomplete and the resulting mixture was purified by reverse phase HPLCusing C18 column, eluted with 20-90% acetonitrile-H₂O containing 0.05%trifluoroacetic acid (TFA). The fractions containing the desired productwere pooled and concentrated to obtain (1R,3S,4S)-tert-butyl3-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-3-(((S)-1-methoxy-1-oxo-3-phenylpropan-2-yl)amino)-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)-2-azabicyclo[2.2.1]heptane-2-carboxylate:MS m/z 856.6 (M+1); retention time 1.67 minutes.

Step 4:

The product obtained in step 3 was dissolved in dichloromethane (DCM)(2.0 mL) and treated with TFA (0.5 mL). The reaction mixture was stirredat room temperature for 1 hour. LCMS analysis showed the reaction wascomplete. The reaction mixture was concentrated by rotary evaporator togive (S)-Methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((1R,3S,4S)-2-azabicyclo[2.2.1]heptane-3-carboxamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoateas a TFA salt: MS m/z 756.6 (M+1); retention time 1.22 minutes.

Step 5:

In a 25 mL round bottom flask were added (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((1R,3S,4S)-2-azabicyclo[2.2.1]heptane-3-carboxamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoateTFA salt (38.4 mg, 0.044 mmol), LiOH monohydrate (50.0 mg, 1.19 mmol)and a solvent mixture of MeOH—H₂O (2:1, 4.0 mL). The mixture was stirredat room temperature for 60 hours. The LC-MS analysis indicated thereaction was complete. The reaction mixture was concentrated andpurified by reverse phase HPLC, C18 column, eluted with acetonitrile-H₂O(10-70%) containing 0.05% TFA. The fractions containing the desiredproduct were combined and concentrated to give(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((1R,3S,4S)-2-azabicyclo[2.2.1]heptane-3-carboxamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid as a TFA salt, MS m/z 742.5 (M+1). Retention time 1.15 minutes.

Step 6:

To a solution of 3-(2-(maleimido)ethoxy)propanoic acid (2.2 mg, 0.010mmol) in DMF (1 ml) were added HATU (3.7 mg, 0.0098 mmol) and DIEA (3.6mg, 0.028 mmol). The reaction was stirred for 5 min, and then(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((1R,3S,4S)-2-azabicyclo[2.2.1]heptane-3-carboxamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid (8 mg, 0.0093 mmol) in DMF (0.5 ml) was added. The reaction mixturewas stirred at rt for 1 h and then concentrated and purified bypreparative HPLC (10-60% acetonitrile-H₂O containing 0.05% TFA) toobtain(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((1R,3S,4S)-2-(3-(2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoyl)-2-azabicyclo[2.2.1]heptane-3-carboxamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid (C1). MS m/z 937.5 (M+H). Retention time 1.138 min.

Example 2:(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((1R,3S,4S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-2-azabicyclo[2.2.1]heptane-3-carboxamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid (C2)

(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((1R,3S,4S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-2-azabicyclo[2.2.1]heptane-3-carboxamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid (2) was made according to the method in Example 1, except in step 66-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (EMCA)(1.2 mg,0.0058 mmol) in DMF (1.0 mL) was used in place of3-(2-(maleimido)ethoxy)propanoic acid.(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((1R,3S,4S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-2-azabicyclo[2.2.1]heptane-3-carboxamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid (2) MS m/z 935.6 (M+1). Retention time 1.17 minutes.

Example 3:(1R,3S,4S)—N—((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-(4-((2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)propylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-2-methyl-2-azabicyclo[2.2.1]heptane-3-carboxamide(C3)

Step 1:

To a stirred solution of sodium azide (3.50 g, 53.8 mmol) in water (25mL) was added a solution of 1,3-propane sulfone (6.10 g, 50.0 mmol) inacetone (25 mL). The reaction mixture was stirred at room temperaturefor 24 hours and concentrated to dryness. The resulting solid wassuspended in diethyl ether (100 mL) and stirred at reflux for 1 hour.The suspension was cooled to room temperature and the solid wascollected by filtration, washed with acetone and diethyl ether, anddried under vacuum, affording 3-azido-1-propanesulfonic acid. MS m/z188.1 (M+1). ¹H NMR (400 MHz, CD₃OD): δ 3.47 (t, J=6.8 Hz, 2H), 2.87 (t,J=7.6 Hz, 2H), 2.07-2.00 (m, 2H).

Step 2:

3-Azido-1-propanesulfonic acid (2.07 g, 13.0 mmol) was suspended intoluene. PCl₅ (2.61 g, 13.0 mmol) was added. The mixture was heated atreflux for 3 hours. The reaction mixture was cooled to room temperature,and filtered to remove insolubles. The filter cake was washed with DCM.The combined filtrates were concentrated to give3-azidopropane-1-sulfonyl chloride as a dark yellow oil, which was usedin the next step without further purification.

Step 3:

To NH₄OH (5 mL) cooled at 000° C. was added 3-azidopropane-1-sulfonylchloride (1.75 g, 9.53 mmol). After 10 minutes, the reaction mixture waswarmed to room temperature and stirred at the same temperature for 3hours. The oily mixture became clear. The reaction mixture was extractedwith EtOAc three times. The organic phase was washed with brine, driedover anhydrous MgSO₄, and concentrated. The residual solvent was furtherremoved under high vacuum for 18 hours to give3-azidopropane-1-sulfonamide. MS m/z 187.1 (M+1). ¹H NMR (400 MHz,CDCl₃): δ 4.83 (s, 2H), 3.51 (t, J=6.4 Hz, 2H), 3.23 (t, J=7.6 Hz, 2H),2.17-2.10 (m, 2H).

Step 4:

(S)-2-((tert-Butoxycarbonyl)amino)-3-phenylpropanoic acid (100 mg, 0.38mmol) was dissolved in DMF (4 mL), followed by addition of DIEA (0.395mL, 2.26 mmol) and HATU (358 mg, 0.940 mmol). After 15 minutes,3-azidopropane-1-sulfonamide (186 mg, 1.13 mmol) was added. The reactionmixture was stirred for 2 hours at which time LCMS analysis indicatedthe completion of the reaction. The resulting mixture was then purifiedby reverse phase HPLC using C18 column, eluted with 10-90%acetonitrile-H₂O containing 0.05% TFA. The fractions containing thedesired product were pooled and lyophilized to obtain (S)-tert-butyl(1-(3-azidopropylsulfonamido)-1-oxo-3-phenylpropan-2-yl)carbamate. MSm/z 312.1 (M+1-Boc). Retention time 1.15 minutes. The product thusobtained (72.4 mg. 0.176 mmol) was dissolved in 3M methanolic HCl (5mL). The solvent was removed under reduced pressure. The residue wastaken up in acetonitrile and H₂O and lyophilized to give(S)-2-amino-N-((3-azidopropyl)sulfonyl)-3-phenylpropanamide as a pinkishyellowish solid. MS m/z 312.1 (M+1). ¹H NMR (400 MHz, CD₃OD): δ7.42-7.31 (m, 5H), 4.16-4.13 (m, 1H), 3.51-3.47 (m, 4H), 3.32-3.26 (m,1H), 3.13-3.08 (m, 1H), 2.00-1.94 (m, 2H).

Step 5:

To Boc-Val-Dil-Dap-OH (195 mg, 0.34 mmol) dissolved in DMF (4 mL) wereadded DIEA (132 mg, 1.02 mmol) and HATU (108 mg, 0.28 mmol). Thereaction mixture was stirred for 15 minutes at room temperature before(S)-2-amino-N-((3-azidopropyl)sulfonyl)-3-phenylpropanamide (59.2 mg,0.17 mmol) was added. The reaction mixture was stirred for additional 2hours at room temperature. And then purified by reverse-phase HPLC toafford the desired product (95 mg, 65% yield, MS m/z 865.4 (M+1),Retention time 1.43 minutes). The product was dissolved in 3M HCl inMeOH (3 mL). Solvents were removed under vacuum. Then acetonitrile andH₂O were added to the residue and the solution was lyophilized to obtainthe desired product,(S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-azidopropylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-2-amino-3-methyl-1-oxobutane.MS m/z 765.4 (M+1). Retention time 1.04 minutes.

Step 6:

To(1R,3S,4S)-2-(tert-butoxycarbonyl)-2-azabicyclo[2.2.1]heptane-3-carboxylicacid (16.5 mg, 0.068 mmol) in DMF (2.0 mL) were added DIEA (17.6 mg,0.137 mmol) and HATU (21.6 mg, 0.057 mmol). The reaction mixture wasstirred at room temperature for 10 minutes before(S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-azidopropylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-2-amino-3-methyl-1-oxobutane(20 mg, TFA salt, 0.023 mmol) was added. The reaction mixture wasstirred for 2 hours at room temperature at which time LCMS analysisindicated the completion of the reaction. The resulting mixture was thenpurified by reverse phase HPLC using C18 column, eluted with 10-90%ACN-H₂O containing 0.05% TFA. The fractions containing the desiredproduct were pooled and lyophilized to obtain(1R,3S,4S)—N—((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-azidopropylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-2-(tert-butoxycarbonyl)-2-azabicyclo[2.2.1]heptane-3-carboxamide.MS m/z 988.5 (M+1). Retention time 1.51 minutes. The product thusobtained (9.4 mg. 0.0095 mmol) was dissolved in methanolic HCl (3M, 2.0mL). The solvent was removed slowly under reduced pressure. The residuewas dissolved in acetonitrile and H₂O and lyophilized to give(1R,3S,4S)—N—((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-Azidopropylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-2-azabicyclo[2.2.1]heptane-3-carboxamideas a HCl salt. MS m/z 888.5 (M+1). Retention time 1.10 minutes.

Step 7:

(1R,3S,4S)—N—((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-Azidopropylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-2-azabicyclo[2.2.1]heptane-3-carboxamide(8.8 mg, 0.0099 mmol) was dissolved in MeOH (2.0 mL). Paraformaldehyde(10.1 mg, 0.337 mmol) and acetic acid (0.0102 mL) were added, followedby sodium cyanoborohydride (21.2 mg, 0.337 mmol). The reaction mixturewas heated at 50° C. with stirring for 1 hour. Additionalparaformaldehyde (10.1 mg, 0.337 mmol), acetic acid (0.0102 mL) andsodium cyanoborohydride (21.2 mg, 0.337 mmol) were added. After 1 hourat 50° C., LCMS analysis indicated the completion of the reaction. Theresulting mixture was then purified by reverse phase HPLC using C18column, eluted with 10-90% ACN-H₂O containing 0.05% TFA. The fractionscontaining the desired product were pooled and lyophilized to obtain(1R,3S,4S)—N—((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-Azidopropylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-2-methyl-2-azabicyclo[2.2.1]heptane-3-carboxamide.MS m/z 902.5 (M+1). Retention time 1.12 minutes.

Step 8:

A solution of(1R,3S,4S)—N—((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-azidopropylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-2-methyl-2-azabicyclo[2.2.1]heptane-3-carboxamide(5.2 mg, 0.0058 mmol), 1-(prop-2-yn-1-yl)-1H-pyrrole-2,5-dione (1.56 mg,0.012 mmol) and CuSO4 (0.7 mg, 0.004 mmol) in DMF (2.0 mL) and H₂O (0.5mL) was treated with L-ascorbic acid sodium salt (2.5 mg, 0.014 mmol)and stirred at room temperature for 2 hours. Additional CuSO4 (0.7 mg,0.004 mmol) and L-ascorbic acid sodium salt (2.5 mg, 0.014 mmol) wereadded. After additional 2 hours at room temperature, LCMS analysisindicated the completion of the reaction. The resulting mixture was thenpurified by reverse phase HPLC using C18 column, eluted with 10-90%acetonitrile-H₂O containing 0.05% TFA. The fractions containing thedesired product were pooled and lyophilized to obtain(1R,3S,4S)—N—((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-(4-((2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)propylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-2-methyl-2-azabicyclo[2.2.1]heptane-3-carboxamide(C3). MS m/z 1037.4 (M+1). Retention time 1.00 minutes.

Example 4: Synthesis of(S)-2-((bis(dimethylamino)methylene)amino)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)propylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutane(C4)

Step 1:

To a stirred solution of sodium azide (3.5 g, 54 mmol) in water (25 ml)was added a solution of 1,3-propane sulfone (6.1 g, 50 mmol) in acetone(25 ml). The reaction mixture was stirred at rt for 24 h, andconcentrated. The resulting solid was suspended in diethyl ether (100ml) and stirred at reflux for 1 h. The suspension was cooled to rt. Thesolid was collected by filtration, washed with acetone and diethylether, and dried under vacuum, affording of 3-azido-1-propanesulfonicacid. MS m/z 188.1 (M+23). ¹H NMR (400 MHz, CD₃OD): δ 3.47 (t, J=6.8 Hz,2H), 2.87 (t, J=7.6 Hz, 2H), 2.07-2.00 (m, 2H).

Step 2:

3-Azido-1-propanesulfonic acid (2.07 g, 13 mmol) was suspended intoluene. PCl₅ (2.61 g, 13 mmol) was added. The mixture was heated atreflux for 3 h. The reaction was cooled to rt. Insoluble matters wereremoved by filtration, and washed with DCM. The combined filtrate wasconcentrated to give 3-azidopropane-1-sulfonyl chloride as ayellow-brown oil, which was used in the next step without furtherpurification.

Step 3:

NH₄OH (28%, 5 mL) was cooled to 0° C. 3-azidopropane-1-sulfonyl chloride(1.75 g, 9.53 mmol) was added. After 10 min, the reaction was warmed tort, and then was stirred for 3 hours at rt. The two phases becamehomogeneous. The reaction mixture was extracted with EtOAc three times.The combined organic phases was washed with brine, dried over MgSO₄, andconcentrated on a rotary evaporator followed by high vacuum for 18 h togive 3-azidopropane-1-sulfonamide. MS m/z 187.1 (M+23). ¹H NMR (400 MHz,CDCl₃): δ 4.83 (s, 2H), 3.51 (t, J=6.4 Hz, 2H), 3.23 (t, J=7.6 Hz, 2H),2.17-2.10 (m, 2H).

Step 4:

(S)-2-((tert-Butoxycarbonyl)amino)-3-phenylpropanoic acid (100 mg, 0.38mmol) was dissolved in DMF (4 mL). DIEA (0.395 mL, 2.26 mmol) and HATU(358 mg, 0.94 mmol) were added. After 15 min,3-azidopropane-1-sulfonamide (186 mg, 1.13 mmol) was added. The reactionwas stirred for 2 h. LCMS indicated a completion of the reaction. Thereaction mixture was purified by preparative HPLC using a 10-90%gradient to obtain (S)-tert-butyl(1-(3-azidopropylsulfonamido)-1-oxo-3-phenylpropan-2-yl)carbamate. MSm/z 312.1 (M+1-Boc). Retention time 1.15 min. The product thus obtained(72.4 mg. 0.176 mmol) was dissolved in methanolic HCl (3 M, 5 mL). Thesolvent was removed by evaporation. The residue was lyophilized fromacetonitrile and H₂O to give(S)-2-amino-N-((3-azidopropyl)sulfonyl)-3-phenylpropanamide as a pinkishyellowish solid. MS m/z 312.1 (M+1)¹H NMR (400 MHz, CD₃OD): δ 7.42-7.31(m, 5H), 4.16-4.13 (m, 1H), 3.51-3.47 (m, 4H), 3.32-3.26 (m, 1H),3.13-3.08 (m, 1H), 2.00-1.94 (m, 2H).

Step 5:

To Boc-Val-Dil-Dap-OH (195 mg, 0.3 4 mmol) in DMF (4 mL) were added DIEA(132 mg, 1.02 mmol) and HATU (108 mg, 0.28 mmol). It was stirred 15 minat rt. (S)-2-amino-N-((3-azidopropyl)sulfonyl)-3-phenylpropanamide (59.2mg, 0.17 mmol) was added. The reaction was stirred for 2 h at rt. Thecrude material was purified by preparative HPLC to afford the desiredproduct (95 mg, 65% yield, MS m/z 865.4 (M+1), Retention time 1.43minutes). The product was dissolved in 3M HCl in MeOH (3 mL). Solventswere removed by evaporation. The residue was lyophilized fromacetonitle-water to obtain(S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-azidopropylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-2-amino-3-methyl-1-oxobutane,

as an HCl salt, MS m/z 765.4 (M+1), retention time 1.04 min.

Step 6:

To(S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-azidopropylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-2-amino-3-methyl-1-oxobutaneHCl salt (20 mg, 0.025 mmol) in DMF (2 mL) were added DIEA (0.024 mL,0.14 mmol) and HATU (21.6 mg, 0.057 mmol). The reaction was stirred atrt for 2 h. LCMS indicated completion of the reaction. The resultingmixture was then purified by preparative HPLC using a 10-90% gradient toobtain(S)-2-((bis(dimethylamino)methylene)amino)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-azidopropylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutaneas a TFA salt. MS m/z 863.5 (M+1). Retention time 1.169 min.

Step 7:

(S)-2-((Bis(dimethylamino)methylene)amino)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-azidopropylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutaneTFA salt (87.4 mg, 0.089 mmol) and1-(prop-2-yn-1-yl)-1H-pyrrole-2,5-dione (24.2 mg, 0.0179 mmol) weresuspended in 3.0 mL each of t-BuOH and water. The reaction vessel wasfilled with N₂ by vacuum-fill cycle with N₂ five times. Degassedsolutions of sodium L-ascorbate (17.7 mg, 0.089 mmol) in H₂O (2.4 ml)and CuSO₄ (2.86 mg, 0.018 mmol) in H₂O (0.6 ml) were added successivelyand the reaction was stirred at rt for 5 h. LCMS indicated completion ofthe reaction. The crude material was purified by preparative HPLC usinga 20-45% gradient to obtain(S)-2-((bis(dimethylamino)methylene)amino)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)—N-1-(3-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)propylsulfonamido)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutane(C4) as a TFA salt. MS m/z 998.5 (M+1). Retention time 1.014 min.

Example 5: Synthesis of6′O-methyl-7′C-((23-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21-heptaoxatricosanthio)methyl)-α-Amanitin(C5),7′C-((23-azido-3,6,9,12,15,18,21-heptaoxatricosanthio)methyl)-α-Amanitin(A-3) and6′O-methyl-7′C-((23-azido-3,6,9,12,15,18,21-heptaoxatricosanthio)methyl)-α-Amanitin(A4)

Step 1:

Formaldehyde (0.035 mL, 0.44 mmol) and23-azido-3,6,9,12,15,18,21-heptaoxatricosane-1-thiol (35 mg, 0.11 mmol)were added to a solution of α-Amanitin (20 mg, 0.022 mmol) in MeOH (2mL). Triethylamine (1.2 mL, 8.7 mmol) and acetic acid (0.25 mL, 4.4mmol) were added to the reaction mixture and flushed with N₂ gas threetimes. The reaction mixture was stirred at 40° C. for 2 days. Afterconcentration in vacuo, the residue was then purified by HPLC andlyophilized to give7′C-((23-azido-3,6,9,12,15,18,21-heptaoxatricosanthio)methyl)-α-Amanitin.MS (m+1)=1342.4, HPLC Peak RT=0.834 min, 1H-NMR (MeOD, 500 MHz) δ 10.65(s, 1H), 8.81 (m, 1H), 8.59 (d, 1H, J=2.0 Hz), 8.45 (m, 2H), 8.33 (s,1H), 8.14 (d, 1H, J=10.5 Hz), 8.00 (d, 1H, J=12.0 Hz), 7.90 (d, 1H,J=11.0 Hz), 7.67 (s, 1H), 7.48 (d, 1H, J=11.0 Hz), 6.69 (d, 1H, J=10.5Hz), 5.25 (m, 1H), 5.12 (m, 1H), 4.74 (bs, 1H), 4.61 (dd, 1H, J=6.5 and12.0 Hz), 4.51 (m, 2H), 4.29 (dd, 1H, J=10.5 and 23.0 Hz), 4.09 (m, 3H),3.92 (m, 1H), 3.38-3.73 (m, 43H), 3.29 (m, 2H), 3.21 (m, 1H), 3.06 (m,1H), 3.12 (m, 1H), 2.91 (m, 1H), 2.56 (m, 2H), 2.39 (m, 2H), 2.00 (m,1H), 1.60 (m, 2H), 1.15 (m, 1H), 0.94 (d, 3H, J=9.0 Hz), 0.85 (m, 6H).

Step 2:

7′C-((23-azido-3,6,9,12,15,18,21-heptaoxatricosanthio)methyl)-α-Amanitin(14.0 mg, 0.011 mmol) and DMSO (1 mL) were treated with methyliodide(0.0007 mL) and K2CO3 (1.5 mg) at rt and stirred at rt for 1 h.Additional methyliodide (0.0007 mL) and K2CO3 (1.5 mg) were added at rtand stirred at rt for 2 h. Additional methyliodide (0.0007 mL) and K2CO3(1.5 mg) at rt and stirred at rt for 2 h, again. The reaction mixturewas then purified by RP-C18 ISCO and lyophilized to give6′O-methyl-7′C-((23-azido-3,6,9,12,15,18,21-heptaoxatricosanthio)methyl)-α-Amanitin.MS (m+2/2)=679.0, HPLC Peak RT=0.887 min, 1H-NMR (MeOD, 500 MHz) δ 10.75(s, 1H), 8.83 (m, 1H), 8.64 (d, 1H, J=2.0 Hz), 8.52 (d, 1H, J=10.0 Hz),8.47 (d, 1H, J=3.5 Hz), 8.36 (s, 1H), 8.18 (d, 1H, J=8.5 Hz), 8.05 (d,1H, J=9.5 Hz), 7.96 (d, 1H, J=9.0 Hz), 7.70 (d, 1H, J=9.0 Hz), 7.69 (s,1H), 6.98 (d, 1H, J=9.0 Hz), 5.33 (m, 1H), 5.18 (m, 1H), 4.80 (bs, 1H),4.68 (dd, 1H, J=5.5 and 9.5 Hz), 4.56 (m, 2H), 4.35 (dd, 1H, J=9.0 and18.5 Hz), 4.10˜4.21 (m, 3H), 3.97 (m, 1H), 3.92 (s, 3H), 3.45˜3.79 (m,42H), 3.35˜3.44 (m, 3H), 3.11 (m, 1H), 2.96 (m, 1H), 2.61 (m, 2H), 2.44(m, 2H), 2.06 (m, 1H), 1.65 (m, 2H), 1.21 (m, 1H), 0.99 (d, 3H, J=7.0Hz), 0.90 (m, 6H).

Step 3:

6′O-methyl-7′C-((23-azido-3,6,9,12,15,18,21-heptaoxatricosanthio)methyl)-α-Amanitin(8 mg, 0.006 mmol) and 1-(prop-2-yn-1-yl)-1H-pyrrole-2,5-dione (2 mg,0.012 mmol) were added to t-butanol (0.5 mL) and the reaction mixturewas flushed with N₂ gas five times. L-Ascorbic acid sodium salt (1 mg,0.006 mmol), CuSO₄ (0.2 mg, 0.0012 mmol) and 0.5 mL of H₂O were thenadded. The reaction mixture was flushed with N₂ gas five times andstirred at rt for 4 h, and then purified by RP-C18 ISCO to give6′O-methyl-7′C-((23-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21-heptaoxatricosanthio)methyl)-α-Amanitin(C5). MS (m+2/2)=746.5, HPLC Peak RT=0.850 min, 1H-NMR (MeOD, 500 MHz) δ10.74 (s, 1H), 8.83 (m, 1H), 8.63 (d, 1H, J=2.0 Hz), 8.51 (d, 1H, J=10.0Hz), 8.47 (d, 1H, J=3.5 Hz), 8.36 (s, 1H), 8.17 (d, 1H, J=8.5 Hz), 8.04(d, 1H, J=10.0 Hz), 7.96 (d, 1H, J=9.5 Hz), 7.94 (s, 1H), 7.69 (d, 1H,J=9.0 Hz), 6.97 (d, 1H, J=9.0 Hz), 6.83 (s, 2H), 5.34 (m, 1H), 5.17 (m,1H), 4.79 (bs, 1H), 4.75 (s, 2H), 4.68 (dd, 1H, J=5.0 and 9.5 Hz), 4.56(m, 2H), 4.52 (t, 1H, J=5.0 Hz), 4.34 (dd, 1H, J=9.0 and 18.5 Hz),4.08˜4.20 (m, 3H), 3.97 (m, 1H), 3.91 (s, 3H), 3.39˜3.78 (m, 38H), 3.10(m, 1H), 2.94 (dd, 1H, J=14.0 and 15.0 Hz), 2.61 (m, 2H), 2.41 (m, 2H),2.05 (m, 1H), 1.57-1.68 (m, 2H), 1.20 (m, 1H), 0.99 (d, 3H, J=7.0 Hz),0.91 (m, 6H).

Example 6: Synthesis of6′O-methyl-7′C-((4-(3-(23-((4-maleimido)methyl-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21-heptaoxatricosyl)ureido)butylthio)methyl)-α-Amanitin(C6)

Step 1:

Formaldehyde (0.027 mL, 0.33 mmol) and tert-butyl(4-mercaptobutyl)carbamate (i-7) (34 mg, 0.16 mmol) were added to asolution of α-Amanitin (A) (15 mg, 0.016 mmol) in MeOH (5 mL) andtriethylamine (0.46 mL, 3.26 mmol) in a 40 mL vial, and the reactionmixture was stirred at 40° C. for 3 days. After concentration in vacuo,the residue was dissolved in 2 mL of MeOH and 408 μL of 2M oftrimethylsilyl diazomethane in diethyl ether was added and the mixturewas stirred for 2 h at rt. Then another 408 μL of 2M of trimethylsilyldiazomethane in diethyl ether was added and stirred at rt for 2 h. Thereaction mixture was purified by HPLC and lyophilized to give6′O-methyl-7′C-((4-t-butoxycarbonylaminobutylthio)methyl)-α-Amanitin(A-4),

MS (m+2-boc/2)=525.8, HPLC Peak RT=0.936 min, 1H-NMR (MeOD-d4, 400 MHz)δ 10.78 (s, 1H), 8.84 (m, 1H), 8.59 (d, 1H, J=2.4 Hz), 8.48 (s, 1H),8.46 (d, 1H, J=14.4 Hz), 8.35 (s, 1H), 8.15 (d, 1H, J=8.8 Hz), 8.01 (d,1H, J=10.0 Hz), 7.92 (d, 1H, J=8.8 Hz), 7.69 (s, 1H), 7.63 (d, 1H, J=8.8Hz), 6.92 (d, 1H, J=8.8 Hz), 5.28 (m, 1H), 5.13 (m, 1H), 4.73 (bs, 1H),4.61 (dd, 1H, J=5.6 and 8.4 Hz), 4.51 (m, 2H), 4.30 (dd, 1H, J=8.8 and18.4 Hz), 4.12 (m, 1H), 4.04 (d, 1H, J=13.2 Hz), 3.94 (d, 1H, J=13.2Hz), 3.92 (m, 1H), 3.86 (s, 3H), 3.35˜3.75 (m, 14H), 3.05 (m, 1H), 2.92(m, 3H), 2.49 (m, 4H), 2.00 (m, 1H), 1.39˜1.65 (m, 8H), 1.37 (s, 9H),1.15 (m, 1H), 0.93 (d, 3H, J=7.2 Hz), 0.84 (m, 6H).

Step 2:

TFA (1 mL) was added to 8 mg of compound (A-4) in a 40 mL vial and theresulting solution was allowed to stand at rt for 2 min and thenconcentrated under vacuum to give6′O-methyl-7′C-((4-aminobutylthio)methyl)-α-Amanitin (A-5),

which was used without further purification. MS (m+1)=1050.4, HPLC PeakRT=0.635 min, 1H-NMR (MeOD-d4, 400 MHz) δ 8.87 (m, 1H), 8.58 (d, 1H,J=2.4 Hz), 8.48 (d, 1H, J=10.4 Hz), 8.44 (d, 1H, J=1.6 Hz), 8.16 (d, 1H,J=8.4 Hz), 7.97 (d, 1H, J=9.6 Hz), 7.94 (d, 1H, J=9.2 Hz), 7.62 (d, 1H,J=8.8 Hz), 6.92 (d, 1H, J=9.2 Hz), 5.25 (m, 1H), 5.13 (m, 1H), 4.73 (m,1H), 4.60 (dd, 1H, J=5.6 and 9.2 Hz), 4.49 (m, 2H), 4.28 (dd, 1H, J=8.8and 18.4 Hz), 4.12 (m, 1H), 4.04 (d, 1H, J=13.2 Hz), 3.99 (s, 2H), 3.92(m, 1H), 3.86 (s, 3H), 3.83 (s, 1H), 3.60˜3.72 (m, 4H), 3.30˜3.60 (m,10H), 3.00˜3.20 (m, 2H), 2.90 (m, 1H), 2.76 (m, 2H), 2.00 (m, 1H),1.50˜1.75 (m, 7H), 1.16 (d, 1H, J=5.6 Hz), 1.24 (d, 2H, J=7.6 Hz), 1.15(m, 1H), 0.94 (d, 3H, J=6.8 Hz), 0.85 (m, 6H).

Step 3:

Triethylamine (3 μL, 18 μmol) was added to a solution of compound (A-5)(7.5 mg, 7 μmol) and 4-nitrophenyl(23-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21-heptaoxatricosyl)carbamate(5.0 mg, 7 μmol) in DMF (1 mL) and the reaction mixture was stirred atrt for 2 h, purified by HPLC and lyophilized to give6′O-methyl-7′C-((4-(3-(23-((4-maleimido)methyl-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21-heptaoxatricosyl)ureido)butylthio)methyl)-α-Amanitin(C6). MS (m+2/2)=803.5, HPLC Peak RT=0.834 min, 1H-NMR (MeOD-d4, 400MHz) δ 10.76 (s, 1H), 8.84 (m, 1H), 8.59 (d, 1H, J=2.0 Hz), 8.49 (s,1H), 8.47 (d, 1H, J=8.0 Hz), 8.15 (d, 1H, J=8.4 Hz), 8.01 (d, 1H, J=9.6Hz), 7.92 (d, 1H, J=8.8 Hz), 7.90 (s, 1H), 7.63 (d, 1H, J=8.8 Hz), 6.92(d, 1H, J=9.2 Hz), 6.79 (s, 2H), 5.28 (m, 1H), 5.13 (m, 1H), 4.74 (m,1H), 4.71 (s, 2H), 4.62 (dd, 1H, J=5.2 and 9.6 Hz), 4.49 (m, 4H), 4.30(dd, 1H, J=8.8 and 18.4 Hz), 4.14 (m, 1H), 4.04 (d, 1H, J=13.2 Hz), 3.94(d, 1H, J=13.2 Hz), 3.92 (m, 1H), 3.85 (s, 3H), 3.80 (t, 2H, J=4.8 Hz),3.35˜3.75 (m, 38H), 2.90˜3.10 (m, 4H), 2.92 (m, 1H), 2.40 (m, 4H), 2.01(m, 1H), 1.38˜1.65 (m, 6H), 1.15 (m, 1H), 0.94 (d, 3H, J=6.8 Hz), 0.85(m, 6H).

Example 7: Synthesis of Tetrafluorophenyl ester of6′O-methyl-7′C-((4-(3-(carboxy)propanecarboxamido)butylthio)methyl)-α-Amanitin(C7)

(A-5) (5 mg, 5 μmol) and DMF (1 mL) were combined in a 40 mL vial togive a clear solution. bis(2,3,5,6-tetrafluorophenyl) glutarate (2 mg, 5μmol) and DIEA (4 μL, 20 μmol) were added. After the reaction mixturewas stirred at rt for 2 h, the reaction mixture was purified by HPLC togive Tetrafluorophenyl ester of6′O-methyl-7′C-((4-(3-(carboxy)propanecarboxamido)butylthio)methyl)-α-Amanitin(C-7). MS (m+1)=1313.3, HPLC Peak RT=0.996 min, 1H-NMR (MeOD, 400 MHz) δ10.78 (s, 1H), 8.85 (m, 1H), 8.59 (s, 1H), 8.49 (s, 1H), 8.47 (d, 1H,J=10.0 Hz), 8.35 (bs, 1H), 8.15 (d, 1H, J=8.0 Hz), 8.01 (d, 1H, J=9.6Hz), 7.93 (d, 1H, J=8.8 Hz), 7.69 (bs, 1H), 7.63 (d, 1H, J=8.8 Hz), 7.36(m, 1H), 6.92 (d, 1H, J=8.8 Hz), 5.27 (m, 1H), 5.14 (m, 1H), 4.75 (bs,1H), 4.61 (dd, 1H, J=5.2 and 9.6 Hz), 4.51 (m, 2H), 4.30 (dd, 1H, J=8.4and 18.0 Hz), 4.12 (m, 1H), 4.00 (d, 1H, J=13.2 Hz), 3.95 (d, 1H, J=13.2Hz), 3.91 (m, 1H), 3.85 (s, 3H), 3.34-3.70 (m, 9H), 3.08 (m, 4H), 2.91(m, 1H), 2.73 (t, 2H, J=14.4 Hz), 1.98 (t, 2H, J=7.6 Hz), 1.92-2.04 (m,1H), 1.40˜1.60 (m, 6H), 1.16 (m, 1H), 0.94 (d, 3H, J=6.8 Hz), 0.87 (m,6H).

Other compounds of Formula (A), Formula (B), Formula (A-1), Formula(A-2), Formula (A-3), Formula (B-1), Formula (A-1a), Formula (A-2a),Formula (A-3a) or Formula (B-1a) can be made using the methods ofExamples 1-7 and appropriate starting materials.

Example 8: Preparation of the Linker Payload MPET.DM4

Analytical Methods

Unless otherwise indicated, the following HPLC and HPLC/MS methods wereused in the preparation of Intermediates and Examples.

LC/MS analysis was performed on an Agilent 1200s1/6140 system.

Column: Waters Acquity HSS T3 C18, 50×2.0, 1.8 um

Mobile Phase: A) H₂O+0.05% TFA; B: Acetonitrile+0.035% TFA

Pump Method:

Time A % B % Flow (mL/min) 0 90 10 0.9 1.35 0 100 0.9 1.36 0 100 0.91.95 0 100 0.9 1.96 90 10 0.9 2.0 90 10 0.9

Detection: UV Diode Array at 190 nm-400 nm

MS Scan: 200-1350 amu

ELSD: 60° C.

MS Parameters:

Polarity Positive Drying Gas 12 Nebulizer Pressure 50 Drying GasTemperature 350 Capillary Voltage 3000

(14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-ylN-(4-((2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethyl)disulfanyl)-4-methylpentanoyl)-N-methyl-L-alaninate

Step 1:

Preparation of(14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-ylN-(4-((2-aminoethyl)disulfanyl)-4-methylpentanoyl)-N-methyl-L-alaninate

To DM4 (480 mg, 0.62 mmol) dissolved in PBS buffer (10.5 mL) andanhydrous THF (21 mL) were added 2-(pyridin-2-yldisulfanyl)ethan-1-amine(151 mg, 0.68 mmol) and DIEA (0.27 mL, 1.54 mmol) at room temperature.The reaction mixture was stirred at room temperature for 30 min andconcentrated in vacuo. The aqueous residue was diluted with CH₃CN (1 mL)and H₂O (2 mL) and purified by reverse phase ISCO, eluted with 10-60%acetonitrile-H₂O containing 0.05% TFA. Fractions containing desiredproduct were lyophilized to obtain desired product (555 mg, 93% yield).¹H NMR (400 MHz, MeOD-d₄) δ ppm 0.83 (s, 3H) 1.21 (d, J=5.0 Hz, 3H) 1.25(s, 3H) 1.28 (s, 3H) 1.30 (d, J=5.0 Hz, 3H) 1.45-1.55 (m, 3H) 1.67 (s,3H) 1.84-1.88 (m, 1H) 1.95-2.01 (m, 1H) 2.14 (dd, J=5.0 and 15.0 Hz, 1H)2.37-2.43 (m, 1H) 2.53-2.59 (m, 1H) 2.64 (dd, J=10.0 and 15.0 Hz, 1H)2.82-2.89 (m, 5H) 2.91 (d, J=10.0 Hz, 1H) 3.16 (dd, J=5.0 and 10.0 Hz,2H) 3.20 (s, 3H) 3.23 (d, J=10.0 Hz, 1H) 3.35 (s, 3H) 3.55 (d, J=5.0 Hz,1H) 3.58 (d, J=10.0 Hz, 1H) 4.15-4.20 (m, 1H) 4.64 (dd, J=5.0 and 10.0Hz, 1H) 5.43 (q, J=5.0 Hz, 2H) 5.66 (dd, J=10.0 and 15.0 Hz, 1H)) 6.58(dd, J=10.0 and 15.0 Hz, 1H) 6.65 (d, J=10.0 Hz, 1H) 6.66 (s, 1H) 7.11(bs, 1H) 7.28 (bs, 1H); MS m/z 855.3 (M+H), Retention time 0.988minutes.

Step 2:

Preparation of(14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-ylN-(4-((2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethyl)disulfanyl)-4-methylpentanoyl)-N-methyl-L-alaninate.

To(14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-ylN-(4-((2-aminoethyl)disulfanyl)-4-methylpentanoyl)-N-methyl-L-alaninate(555 mg, 0.57 mmol) dissolved in anhydrous DMSO (7 mL) were added2,5-dioxopyrrolidin-1-yl3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (171 mg, 0.63 mmol)and DIEA (249 mL, 1.43 mmol) at room temperature. The reaction mixturewas stirred at room temperature for 15 min and neutralized using TFA.The mixture was cooled to 0° C. with iced bath, followed by addition ofCH₃CN (2 mL) and H₂O (7 mL), and then purified by reverse phase ISCO,eluting with 10-70% acetonitrile-H₂O containing 0.05% TFA. Fractionscontaining desired product were lyophilized to obtain desired product(430 mg, 66% yield).). ¹H NMR (400 MHz, CDCl₃) δ ppm 0.81 (s, 3H) 1.23(s, 3H) 1.24 (s, 3H) 1.25 (s, 1H) 1.28 (d, J=5.0 Hz, 3H) 1.31 (d, J=5.0Hz, 3H) 1.43-1.49 (m, 1H) 1.61 (d, J=15.0 Hz, 1H) 1.64 (s, 3H) 1.81-1.87(m, 1H) 1.94-2.01 (m, 1H) 2.19 (dd, J=5.0 and 15.0 Hz, 1H) 2.30-2.36 (m,1H) 2.54 (t, J=5.0 Hz, 2H) 2.61 (dd, J=10.0 and 15.0 Hz, 1H) 2.70 (t,J=5.0 Hz, 2H) 2.88 (s, 3H) 3.00 (d, J=10.0 Hz, 1H) 3.13 (d, J=10.0 Hz,1H) 3.21 (s, 3H) 3.55 (s, 3H) 3.45 (q, J=5.0 Hz, 2H) 3.49 (d, J=5.0 Hz,1H) 3.62 (d, J=10.0 Hz, 1H) 3.83 (t, J=5.0 Hz, 1H) 3.98 (s, 3H) 4.32 (m,1H) 4.80 (dd, J=5.0 and 10.0 Hz, 1H) 5.28 (d, J=5.0 Hz, 1H) 5.66 (dd,J=10.0 and 15.0 Hz, 1H)) 6.22 (bs, 1H) 6.42 (dd, J=10.0 and 15.0 Hz, 1H)6.50 (s, 1H) 6.63 (s, 1H) 6.66 (d, J=10.0 Hz, 1H) 6.70 (s, 2H) 6.83 (s,1H); MS m/z 988.3 (M+H-H₂O), Retention time 1.145 minutes.

3. Conjugation and Preparation of ADCs

Processes for Making Antibody Conjugate of Formula (I)

A general reaction scheme for the formation of conjugates of Formula (I)is shown in Scheme 1 below:

-   -   where: RG₁ is a reactive group, by way of example only a thiol        or amine or ketone, which reacts with a compatible reactive        group, RG₂, attached to the linker-drug moiety thereby        covalently linking antibody fragment, A, to one or more        linker-drug moieties. A non-limiting examples of such reactions        of RG₁ and RG₂ groups is a maleimide (RG₂) reacting with a thiol        (RG₁) to give a succinimide ring, or a hydroxylamine (RG₂)        reacting with a ketone (RG₁) to give an oxime.

A general reaction scheme for the formation of conjugates of Formula(II) is shown in Scheme 2 below:

-   -   where: A₁, A₂, LB, D and n are as defined herein, the        1,3-dihaloacetone is selected from 1,3-dichloroacetone,        1,3-dibromoacetone, and 1,3-diiodoacetone, and the reduction        step is accomplished using a reducing agent selected from        dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine        hydrochloride (TCEP-HCl).

A general reaction scheme for the formation of conjugates of Formula (C)is shown in Scheme 3 below:

where: L₂₀ is -L₁R⁴⁰; R⁴ is -L₁R¹⁴, -L₂R²⁴ or -L₂R³⁴ and RG₁ is areactive group, by way of example only a thiol or amine or ketone, whichreacts with a compatible R¹⁴, R²⁴ or R³⁴ group of a compound of Formula(A) to form a corresponding R⁴⁰ group. By way of example, a maleimidereacting with a thiol to give a succinimide ring, or a hydroxylaminereacting with a ketone to give an oxime. A, R², L₁, L₂, R¹⁴, R²⁴, R³⁴and R⁴⁰ are as defined herein.

A general reaction scheme for the formation of conjugates of Formula(C-1) is shown in Scheme 4 below:

where: L₂₀ is -L₁R⁴⁰; R⁴ is -L₁R¹⁴, -L₂R²⁴ or -L₂R³⁴ and RG₁ is areactive group which reacts with a compatible R¹⁴, R²⁴ or R³⁴ group of acompound of Formula (A-1) to form a corresponding R⁴⁰ group. By way ofexample, a maleimide reacting with a thiol to give a succinimide ring,or a hydroxylamine reacting with a ketone to give an oxime. A, L₁, L₂,R¹⁴, R²⁴, R³⁴ and R⁴⁰ are as defined herein.

A general reaction scheme for the formation of conjugates of Formula(C-1a) is shown in Scheme 5 below:

where: L₂₀ is -L₁R⁴⁰; R⁴ is -L₁R¹⁴, -L₂R²⁴ or -L₂R³⁴ and RG₁ is areactive group, by way of example only a thiol or amine or ketone, whichreacts with a compatible R¹⁴, R²⁴ or R³⁴ group of a compound of Formula(A-1a) to form a corresponding R⁴⁰ group. By way of example, a maleimidereacting with a thiol to give a succinimide ring, or a hydroxylaminereacting with a ketone to give an oxime. A, L₁, L₂, R¹⁴, R²⁴, R³⁴ andR⁴⁰ are as defined herein.

A general reaction scheme for the formation of conjugates of Formula (D)is shown in Scheme 6 below:

where: L₃₀ is -L₅R⁴⁰; R³ is -L₅R¹⁴ and RG₁ is a reactive group which, byway of example only a thiol or amine or ketone, reacts with a compatibleR¹⁴ group of a compound of Formula (A) to form a corresponding R⁴⁰group. By way of example, a maleimide reacting with a thiol to give asuccinimide ring, or a hydroxylamine reacting with a ketone to give anoxime. A, R², Ls, R¹⁴ and R⁴⁰ are as defined herein.

A general reaction scheme for the formation of conjugates of Formula(D-1) is shown in Scheme 7 below:

where: L₃₀ is -L₅R⁴⁰ and RG₁ is a reactive group, by way of example onlya thiol or amine or ketone, which reacts with a compatible R¹⁴ group ofa compound of Formula (A-2) to form a corresponding R⁴⁰ group. By way ofexample, a maleimide reacting with a thiol to give a succinimide ring,or a hydroxylamine reacting with a ketone to give an oxime. A, L₅, R¹⁴and R⁴⁰ are as defined herein.

A general reaction scheme for the formation of conjugates of Formula(D-la) is shown in Scheme 8 below:

where: L₃₀ is -L₅R⁴⁰ and RG₁ is a reactive group, by way of example onlya thiol or amine or ketone, which reacts with a compatible R¹⁴ group ofa compound of Formula (A-2) to form a corresponding R⁴⁰ group. By way ofexample, a maleimide reacting with a thiol to give a succinimide ring,or a hydroxylamine reacting with a ketone to give an oxime. A, L₅, R¹⁴and R⁴⁰ are as defined herein.

A general reaction scheme for the formation of conjugates of Formula(D-2) is shown in Scheme 9 below:

where: L₃₀ is -L₅R⁴⁰ and RG₁ is a reactive group, by way of example onlya thiol or amine or ketone, which reacts with a compatible R¹⁴ group ofa compound of Formula (A-3) to form a corresponding R⁴⁰ group. By way ofexample, a maleimide reacting with a thiol to give a succinimide ring,or a hydroxylamine reacting with a ketone to give an oxime. A, L₅, R¹⁴and R⁴⁰ are as defined herein.

A general reaction scheme for the formation of conjugates of Formula(D-2a) is shown in Scheme 10 below:

where: L₃₀ is -L₅R⁴⁰ and RG₁ is a reactive group, by way of example onlya thiol or amine or ketone, which reacts with a compatible R¹⁴ group ofa compound of Formula (A-3a) to form a corresponding R⁴⁰ group. By wayof example, a maleimide reacting with a thiol to give a succinimidering, or a hydroxylamine reacting with a ketone to give an oxime. A, Ls,R¹⁴ and R⁴⁰ are as defined herein.

A general reaction scheme for the formation of conjugates of Formula (E)is shown in Scheme 11 below:

where: L₄₀ is -L₆R⁴⁰; R⁵⁴ is -L₆R¹⁴, -L₇R²⁴ or -L₇R³⁴ and RG₁ is areactive group, by way of example only a thiol or amine or ketone, whichreacts with a compatible R¹⁴, R²⁴ or R³⁴ group of a compound of Formula(B) to form a corresponding R⁴⁰ group. By way of example, a maleimidereacting with a thiol to give a succinimide ring, or a hydroxylaminereacting with a ketone to give an oxime. A, X, R⁵, R⁶, L₆, L₇, R¹⁴, R²⁴,R³⁴ and R⁴⁰ are as defined herein.

A general reaction scheme for the formation of conjugates of Formula(E-1) is shown in Scheme 12 below:

where: L₄₀ is -L₆R⁴⁰; R⁵⁴ is -L₆R¹⁴, -L₇R²⁴ or -L₇R³⁴ and RG₁ is areactive group, by way of example only a thiol or amine or ketone, whichreacts with a compatible R¹⁴, R²⁴ or R³⁴ group of a compound of Formula(B-1) to form a corresponding R⁴⁰ group. By way of example, a maleimidereacting with a thiol to give a succinimide ring, or a hydroxylaminereacting with a ketone to give an oxime. A, y, R⁵, R⁶, L₆, L₇, R¹⁴, R²⁴,R³⁴ and R⁴⁰ are as defined herein.

A general reaction scheme for the formation of conjugates of Formula(E-1a) is shown in Scheme 13 below:

where: L₄₀ is -L₆R⁴⁰; R⁵⁴ is -L₆R¹⁴, -L₇R²⁴ or -L₇R³⁴ and RG₁ is areactive group, by way of example only a thiol or amine or ketone, whichreacts with a compatible R¹⁴, R²⁴ or R³⁴ group of a compound of Formula(B-1a) to form a corresponding R⁴⁰ group. By way of example, a maleimidereacting with a thiol to give a succinimide ring, or a hydroxylaminereacting with a ketone to give an oxime. A, y, R⁵, R⁶, L₆, L₇, R¹⁴, R²⁴,R³⁴ and R⁴⁰ are as defined herein.

A general reaction scheme for the formation of conjugates comprising amaytansinoid moiety is shown in Scheme 14 below:

where one or more NHS esters of one or more linker-payloads reacts withone or more free amines on A (i.e. (A′-(NH₂)_(y)), thereby forming aconjugate. A is as defined herein and A′ is the portion of A which doesnot include the free amine moiety.

A general reaction scheme for the formation of conjugates comprising amaytansinoid moiety is shown in Scheme 15 below:

where one or more NHS esters of one or more linker-payloads reacts withone or more free amines on A (i.e. (A′-(NH₂)_(y)), thereby forming aconjugate. A is as defined herein and A′ is the portion of A which doesnot include the free amine moiety.

A general reaction scheme for the formation of conjugates comprising amaytansinoid moiety is shown in Scheme 16 below:

where one or more NHS esters of one or more linker-payloads reacts withone or more free amines on A (i.e. (A′-(NH₂)_(y)), thereby forming aconjugate. A is as defined herein and A′ is the portion of A which doesnot include the free amine moiety.

A general reaction scheme for the formation of conjugates comprising amaytansinoid moiety is shown in Scheme 17 below:

where one or more maleimides of one or more linker-payloads reacts withone or more free thiols on A (i.e. (A′-(SH)_(y)), thereby forming aconjugate. A is as defined herein and A′ is the portion of A which doesnot include the free thiol moiety.

4. Characterization and Selection of Desirable Anti-cKIT ADCsDetermination of DAR and Aggregation of the ADCs

DAR value of the cKIT ADC was evaluated by liquid chromatography-massspectrometry (LC-MS). A compound-to-antibody ratio was extrapolated fromLC-MS data for reduced and deglycosylated (when appropriate, i.e. whenFc is included) samples. LC-MS allows quantitation of the average numberof molecules of linker-payload (compound) attached to an antibody in aconjugate sample.

Antibody drug conjugates of the invention were evaluated usinganalytical methods. Such analytical methodology and results candemonstrate that the conjugates have favorable properties, for exampleproperties that would make them easier to manufacture, easier toadminister to patients, more efficacious, and/or potentially safer forpatients. One example is the determination of molecular size by sizeexclusion chromatography (SEC) wherein the amount of desired antibodyspecies in a sample is determined relative to the amount of highmolecular weight contaminants (e.g., dimer, multimer, or aggregatedantibody) or low molecular weight contaminants (e.g., antibodyfragments, degradation products, or individual antibody chains) presentin the sample. In general, it is desirable to have higher amounts ofmonomer and lower amounts of, for example, aggregated antibody due tothe impact of, for example, aggregates on other properties of theantibody sample such as but not limited to clearance rate,immunogenicity, and toxicity. A further example is the determination ofthe hydrophobicity by hydrophobic interaction chromatography (HIC)wherein the hydrophobicity of a sample is assessed relative to a set ofstandard antibodies of known properties. In general, it is desirable tohave low hydrophobicity due to the impact of hydrophobicity on otherproperties of the antibody sample such as but not limited toaggregation, aggregation over time, adherence to surfaces,hepatotoxicity, clearance rates, and pharmacokinetic exposure. SeeDamle, N. K., Nat Biotechnol. 2008; 26(8):884-885; Singh, S. K., PharmRes. 2015; 32(11):3541-71.

Selection of Anti-cKITADCs

To select anti-cKIT ADCs suitable for using in the methods describedherein, an in vitro human hematopoietic stem cell killing assay can beused to screen the anti-cKIT ADCs for their efficacy and potency. Forexample, the methods described in Example 5 can be used to screenanti-cKIT ADCs. Suitable anti-cKIT ADCs can be selected based on EC50,e.g., anti-cKIT ADC with an EC50 less than 500 μg/ml, e.g., less than100 μg/ml, less than 50 μg/ml, less than 10 μg/ml, or less than 5 μg/ml.

Furthermore, it has been reported that cKIT expresses on mast cells, andstem-cell factor (SCF), the ligand of cKIT, induces direct degranulationof rat peritoneal mast cells in vitro and in vivo (Taylor et al.,Immunology. 1995 November; 86(3):427-33). SCF also induces human mastcell degranulation in vivo (Costa et al., J Exp Med. 1996; 183(6):2681-6). To avoid potential detrimental effects caused by mast celldegranulation in transplant recipients, selected cKIT ADCs can be testedfor their ability to induce mast cell degranulation in vitro. Forexample, experiments described in Example 6 can be used to screen cKITADCs, and suitable anti-cKIT ADCs can be selected based on minimal mastcell degranulation, e.g., a baseline corrected O.D. readout of less than0.25, e.g., less than 0.2, less than 0.15, or less than 0.1, in abeta-hexosaminidase release assay.

cKIT Antibody and Antibody Fragments

The present disclosure provides for antibodies or antibody fragments(e.g., antigen binding fragments) that specifically bind to human cKIT.Antibodies or antibody fragments (e.g., antigen binding fragments) ofthe present disclosure include, but are not limited to, the humanmonoclonal antibodies or fragments thereof described below.

In some embodiments, the presently disclosed anti-cKIT antibodies orantibody fragments (e.g., antigen binding fragments) have a reducedability for causing mast cell degranulation, even when cross-linkedand/or multimerized into larger complexes, in comparison to afull-length anti-cKIT antibody. In some embodiments, the anti-cKITantibodies or antibody fragments (e.g., antigen binding fragments)disclosed herein are modified to have reduced ability to induce mastcell degranulation, even when cross-linked and/or multimerized intolarger complexes. For example, the anti-cKIT antibodies or antibodyfragments (e.g., antigen binding fragments) disclosed herein aremodified to have an reduced ability to induce mast cell degranulationthat is, is about, or is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% reduced in comparison to a full-length anti-cKIT antibody, oran F(ab′)₂ or an F(ab)₂ fragment thereof, even when cross-linked and/ormultimerized into larger complexes. In some embodiments, the anti-cKITantibodies or antibody fragments (e.g., antigen binding fragments)disclosed herein may comprise an anti-cKIT Fab or Fab′ fragment. In someembodiments, the anti-cKIT antibodies or antibody fragments (e.g.,antigen binding fragments) disclosed herein may have minimal ability toinduce mast cell degranulation, e.g., a baseline corrected O.D. readoutof less than 0.25, e.g., less than 0.2, less than 0.15, or less than0.1, in a beta-hexosaminidase release assay, even when cross-linkedand/or multimerized into larger complexes.

The antibody drug conjugates provided herein include a humancKIT-binding antibody fragment (e.g., Fab or Fab′). In some embodiments,antibody drug conjugates provided herein include a human or humanizedantibody fragment (e.g., Fab or Fab′) that specifically binds to humancKIT. In some embodiments, antibody drug conjugates provided hereininclude a human or humanized Fab′ that specifically binds to human cKIT.In some embodiments, antibody drug conjugates provided herein include ahuman or humanized Fab that specifically binds to human cKIT.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a VH domain havingan amino acid sequence of any VH domain described in Table 1 (e.g., SEQID NO: 10, 36, 54, 69, 95). Other suitable antibody or antibody fragment(e.g., Fab or Fab′) can include a VH domain that has at least 80, 85,90, 95, 96, 97, 98, or 99 percent sequence identity to any of the VHdomains described in Table 1.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a VH CDR (or HCDR)having an amino acid sequence of any one of the VH CDRs (or HCDR) listedin Table 1. In particular aspects, the present disclosure provides theantibody or antibody fragment (e.g., Fab or Fab′) comprising (oralternatively, consisting of) one, two, three, four, five or more VHCDRs (or HCDR) having an amino acid sequence of any of the VH CDRs (orHCDR) listed in Table 1.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a VL domain havingan amino acid sequence of any VL domain described in Table 1 (e.g., SEQID NO: 23, 47, 82, 108). Other suitable the antibody or antibodyfragment (e.g., Fab or Fab′) can include a VL domain that has at least80, 85, 90, 95, 96, 97, 98, or 99 percent sequence identity to any ofthe VL domains described in Table 1.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a VL CDR (or LCDR)having an amino acid sequence of any one of the VL CDRs (or LCDR) listedin Table 1. In particular aspects, the present disclosure provides theantibody or antibody fragment (e.g., Fab or Fab′) comprising (oralternatively, consisting of) one, two, three, four, five or more VLCDRs (or LCDR) having an amino acid sequence of any of the VL CDRs (orLCDR) listed in Table 1.

Other anti-cKIT antibody or antibody fragment (e.g., Fab or Fab′)disclosed herein include amino acids that have been mutated, yet have atleast 60, 70, 80, 90 or 95 percent sequence identity in the CDR regionswith the CDR regions depicted in the sequences described in Table 1. Insome aspects, it includes mutant amino acid sequences wherein no morethan 1, 2, 3, 4 or 5 amino acids have been mutated in the CDR regionswhen compared with the CDR regions depicted in the sequence described inTable 1.

The present disclosure also provides nucleic acid sequences that encodeVH, VL, the heavy chain, and the light chain of the antibody or antibodyfragment (e.g., Fab or Fab′) that specifically binds to human cKIT. Suchnucleic acid sequences can be optimized for expression in mammaliancells.

TABLE 1Sequences of exemplary anti-cKIT antibodies and antibody fragmentsAnti-cKIT Ab1/Fab1/Fab′1 SEQ ID NO: 1 HCDR1 (Kabat) SYAIS SEQ ID NO: 2HCDR2 (Kabat) VIFPAEGAPGYAQKFQG SEQ ID NO: 3 HCDR3 (Kabat) GGYISDFDVSEQ ID NO: 4 HCDR1 (Chothia) GGTFSSY SEQ ID NO: 5 HCDR2 (Chothia) FPAEGASEQ ID NO: 3 HCDR3 (Chothia) GGYISDFDV SEQ ID NO: 6 HCDR1 (Combined)GGTFSSYAIS SEQ ID NO: 2 HCDR2 (Combined) VIFPAEGAPGYAQKFQG SEQ ID NO: 3HCDR3 (Combined) GGYISDFDV SEQ ID NO: 7 HCDR1 (IMGT) GGTFSSYASEQ ID NO: 8 HCDR2 (IMGT) IFPAEGAP SEQ ID NO: 9 HCDR3 (IMGT) ARGGYISDFDVSEQ ID NO: 10 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGVIFPAEGAPGYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCARGGYISDFDVWGQGTLVTVSS SEQ ID NO: 11 VH DNA CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAAAAAACCGGGCAGCAGCGTGAAAGTTAGCT GCAAAGCATCCGGAGGGACGTTTAGCAGCTATGCGATTAGCTGGGTGCGCCAGGCCCCGGGCC AGGGCCTCGAGTGGATGGGCGTTATCTTCCCGGCTGAAGGCGCTCCGGGTTACGCCCAGAAATT TCAGGGCCGGGTGACCATTACCGCCGATGAAAGCACCAGCACCGCCTATATGGAACTGAGCAGC CTGCGCAGCGAAGATACGGCCGTGTATTATTGCGCGCGTGGTGGTTACATCTCTGACTTCGATG TTTGGGGCCAAGGCACCCTGGTGACTGTTAGC TCASEQ ID NO: 12 Ab HC QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGVIFPAEGAPGYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCARGGYISDFDVWGQGTLVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 13 Ab HC DNA CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAAAAAACCGGGCAGCAGCGTGAAAGTTAGCT GCAAAGCATCCGGAGGGACGTTTAGCAGCTATGCGATTAGCTGGGTGCGCCAGGCCCCGGGCC AGGGCCTCGAGTGGATGGGCGTTATCTTCCCGGCTGAAGGCGCTCCGGGTTACGCCCAGAAATT TCAGGGCCGGGTGACCATTACCGCCGATGAAAGCACCAGCACCGCCTATATGGAACTGAGCAGC CTGCGCAGCGAAGATACGGCCGTGTATTATTGCGCGCGTGGTGGTTACATCTCTGACTTCGATG TTTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCAGCTAGCACCAAGGGCCCCAGCGTGTTCCC CCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGAC TACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCACACCT TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAG CTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACA AGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACT GCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGG ACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAAC AGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAAT ACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAA GGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGA ACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGA GAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAG CTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCT GCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGG CAAG SEQ ID NO: 14 Fab′ HC (EU236)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAI SWVRQAPGQGLEWMGVIFPAEGAPGYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG YISDFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL G SEQ ID NO: 15 Fab′ HC DNACAGGTGCAATTGGTGCAGAGCGGTGCCGAAGT GAAAAAACCGGGCAGCAGCGTGAAAGTTAGCTGCAAAGCATCCGGAGGGACGTTTAGCAGCTAT GCGATTAGCTGGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTGGATGGGCGTTATCTTCCCG GCTGAAGGCGCTCCGGGTTACGCCCAGAAATTTCAGGGCCGGGTGACCATTACCGCCGATGAAA GCACCAGCACCGCCTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGTATTATTG CGCGCGTGGTGGTTACATCTCTGACTTCGATGTTTGGGGCCAAGGCACCCTGGTGACTGTTAGC TCAGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCG GAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAA CTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC AGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGT GAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACC CACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGA SEQ ID NO: 118 Cys Fab- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIHC (EU221)-HC- SWVRQAPGQGLEWMGVIFPAEGAPGYAQKFQG E152C (EU)RVTITADESTSTAYMELSSLRSEDTAVYYCARGG YISDFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD SEQ ID NO: 119 Fab′ HC (EU230)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAI SWVRQAPGQGLEWMGVIFPAEGAPGYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG YISDFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP SEQ ID NO: 120 Fab′ HC (EU232)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAI SWVRQAPGQGLEWMGVIFPAEGAPGYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG YISDFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP SEQ ID NO: 121 Fab′ HC (EU236)-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAI Pro SWVRQAPGQGLEWMGVIFPAEGAPGYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG YISDFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GP SEQ ID NO: 16 LCDR1 (Kabat)RASQSISNYLA SEQ ID NO: 17 LCDR2 (Kabat) DASSLQS SEQ ID NO: 18LCDR3 (Kabat) QQYYYESIT SEQ ID NO: 19 LCDR1 (Chothia) SQSISNYSEQ ID NO: 20 LCDR2 (Chothia) DAS SEQ ID NO: 21 LCDR3 (Chothia) YYYESISEQ ID NO: 16 LCDR1 (Combined) RASQSISNYLA SEQ ID NO: 17LCDR2 (Combined) DASSLQS SEQ ID NO: 18 LCDR3 (Combined) QQYYYESITSEQ ID NO: 22 LCDR1 (IMGT) QSISNY SEQ ID NO: 20 LCDR2 (IMGT) DASSEQ ID NO: 18 LCDR3 (IMGT) QQYYYESIT SEQ ID NO: 23 VL (kappa)DIQMTQSPSSLSASVGDRVTITCRASQSISNYLA WYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYYESITFGQGT KVEIK SEQ ID NO: 24 VL DNAGATATCCAGATGACCCAGAGCCCGAGCAGCCT GAGCGCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGTCTATTTCTAACTACC TGGCTTGGTACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTAATCTACGACGCTTCTTCTCTG CAAAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCTGACCATT AGCTCTCTGCAACCGGAAGACTTTGCGACCTATTATTGCCAGCAGTACTACTACGAATCTATCACC TTTGGCCAGGGCACGAAAGTTGAAATTAAASEQ ID NO: 25 Ab/Fab′ LC (kappa) DIQMTQSPSSLSASVGDRVTITCRASQSISNYLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQYYYESITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC SEQ ID NO: 26 Ab/Fab′LC DNA GATATCCAGATGACCCAGAGCCCGAGCAGCCT GAGCGCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGTCTATTTCTAACTACC TGGCTTGGTACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTAATCTACGACGCTTCTTCTCTG CAAAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCTGACCATT AGCTCTCTGCAACCGGAAGACTTTGCGACCTATTATTGCCAGCAGTACTACTACGAATCTATCACC TTTGGCCAGGGCACGAAAGTTGAAATTAAACGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCC CCCCAGCGACGAGCAGCTGAAGAGTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTA CCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGG AGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAG CAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAG CCCCGTGACCAAGAGCTTCAACAGGGGCGAGT GCSEQ ID NO: 122 Cys Fab-LC-E165C DIQMTQSPSSLSASVGDRVTITCRASQSISNYLA (EU)WYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQYYYESITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTCQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC SEQ ID NO: 123Cys Fab-LC-S114C DIQMTQSPSSLSASVGDRVTITCRASQSISNYLA (EU)WYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQYYYESITFGQGTKVEIKRTVAAPCVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC Anti-cKIT Ab2/Fab2/Fab′2SEQ ID NO: 27 HCDR1 (Kabat) SHALS SEQ ID NO: 28 HCDR2 (Kabat)GIIPSFGTADYAQKFQG SEQ ID NO: 29 HCDR3 (Kabat) GLYDFDY SEQ ID NO: 30HCDR1 (Chothia) GGTFSSH SEQ ID NO: 31 HCDR2 (Chothia) IPSFGTSEQ ID NO: 29 HCDR3 (Chothia) GLYDFDY SEQ ID NO: 32 HCDR1 (Combined)GGTFSSHALS SEQ ID NO: 28 HCDR2 (Combined) GIIPSFGTADYAQKFQGSEQ ID NO: 29 HCDR3 (Combined) GLYDFDY SEQ ID NO: 33 HCDR1 (IMGT)GGTFSSHA SEQ ID NO: 34 HCDR2 (IMGT) IIPSFGTA SEQ ID NO: 35 HCDR3 (IMGT)ARGLYDFDY SEQ ID NO: 36 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHALSWVRQAPGQGLEWMGGIIPSFGTADYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCARGLYDFDYVVGQGTLVTVSS SEQ ID NO: 37 VH DNA CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAAAAAACCGGGCAGCAGCGTGAAAGTTAGCT GCAAAGCATCCGGAGGGACGTTTTCTTCTCATGCTCTGTCTTGGGTGCGCCAGGCCCCGGGCC AGGGCCTCGAGTGGATGGGCGGTATCATCCCGTCTTTCGGCACTGCGGACTACGCCCAGAAATTT CAGGGCCGGGTGACCATTACCGCCGATGAAAGCACCAGCACCGCCTATATGGAACTGAGCAGCC TGCGCAGCGAAGATACGGCCGTGTATTATTGCGCGCGTGGTCTGTACGACTTCGACTACTGGGG CCAAGGCACCCTGGTGACTGTTAGCTCASEQ ID NO: 38 Ab HC QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHALSWVRQAPGQGLEWMGGIIPSFGTADYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCARGLYDFDYVVGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 39 Ab HC DNA CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAAAAAACCGGGCAGCAGCGTGAAAGTTAGCT GCAAAGCATCCGGAGGGACGTTTTCTTCTCATGCTCTGTCTTGGGTGCGCCAGGCCCCGGGCC AGGGCCTCGAGTGGATGGGCGGTATCATCCCGTCTTTCGGCACTGCGGACTACGCCCAGAAATTT CAGGGCCGGGTGACCATTACCGCCGATGAAAGCACCAGCACCGCCTATATGGAACTGAGCAGCC TGCGCAGCGAAGATACGGCCGTGTATTATTGCGCGCGTGGTCTGTACGACTTCGACTACTGGGG CCAAGGCACCCTGGTGACTGTTAGCTCAGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGC CCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTC CCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCG CCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCT GGGAACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGA GTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGG GAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCC CGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCAC CTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAG TGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCC AGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAG GTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGC AACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCT TCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAG CGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG SEQ ID NO: 40 Fab′ HC (EU236)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHA LSWVRQAPGQGLEWMGGIIPSFGTADYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGL YDFDYVVGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL G SEQ ID NO: 41 Fab′ HC DNACAGGTGCAATTGGTGCAGAGCGGTGCCGAAGT GAAAAAACCGGGCAGCAGCGTGAAAGTTAGCTGCAAAGCATCCGGAGGGACGTTTTCTTCTCAT GCTCTGTCTTGGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTGGATGGGCGGTATCATCCCG TCTTTCGGCACTGCGGACTACGCCCAGAAATTTCAGGGCCGGGTGACCATTACCGCCGATGAAAG CACCAGCACCGCCTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGTATTATTGC GCGCGTGGTCTGTACGACTTCGACTACTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCAGCTA GCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTG CTGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGG GGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG AGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACC ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACAC CTGCCCCCCCTGCCCAGCTCCAGAACTGCTGG GASEQ ID NO: 124 Cys Fab- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHA HC (EU221)-HC-LSWVRQAPGQGLEWMGGIIPSFGTADYAQKFQG E152C (EU)RVTITADESTSTAYMELSSLRSEDTAVYYCARGL YDFDYVVGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD SEQ ID NO: 125 Fab′ HC (EU230)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHA LSWVRQAPGQGLEWMGGIIPSFGTADYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGL YDFDYVVGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP SEQ ID NO: 126 Fab′ HC (EU232)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHA LSWVRQAPGQGLEWMGGIIPSFGTADYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGL YDFDYVVGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP SEQ ID NO: 127 Fab′ HC (EU236)-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHA Pro LSWVRQAPGQGLEWMGGIIPSFGTADYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGL YDFDYVVGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GP SEQ ID NO: 42 LCDR1 (Kabat)RASQDISQDLA SEQ ID NO: 17 LCDR2 (Kabat) DASSLQS SEQ ID NO: 43LCDR3 (Kabat) QQYYYLPST SEQ ID NO: 44 LCDR1 (Chothia) SQDISQDSEQ ID NO: 20 LCDR2 (Chothia) DAS SEQ ID NO: 45 LCDR3 (Chothia) YYYLPSSEQ ID NO: 42 LCDR1 (Combined) RASQDISQDLA SEQ ID NO: 17LCDR2 (Combined) DASSLQS SEQ ID NO: 43 LCDR3 (Combined) QQYYYLPSTSEQ ID NO: 46 LCDR1 (IMGT) QDISQD SEQ ID NO: 20 LCDR2 (IMGT) DASSEQ ID NO: 43 LCDR3 (IMGT) QQYYYLPST SEQ ID NO: 47 VL (kappa)DIQMTQSPSSLSASVGDRVTITCRASQDISQDLA WYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYYYLPSTFGQG TKVEIK SEQ ID NO: 48 VL DNAGATATCCAGATGACCCAGAGCCCGAGCAGCCT GAGCGCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGGACATTTCTCAGGAC CTGGCTTGGTACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTAATCTACGACGCTTCTTCTCT GCAAAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCTGACCAT TAGCTCTCTGCAACCGGAAGACTTTGCGGTGTATTATTGCCAGCAGTACTACTACCTGCCGTCTA CCTTTGGCCAGGGCACGAAAGTTGAAATTAAASEQ ID NO: 49 Ab/Fab′ LC (kappa) DIQMTQSPSSLSASVGDRVTITCRASQDISQDLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAVYYCQQYYYLPSTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC SEQ ID NO: 50 Ab/Fab′LC DNA GATATCCAGATGACCCAGAGCCCGAGCAGCCT GAGCGCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGGACATTTCTCAGGAC CTGGCTTGGTACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTAATCTACGACGCTTCTTCTCT GCAAAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCTGACCAT TAGCTCTCTGCAACCGGAAGACTTTGCGGTGTATTATTGCCAGCAGTACTACTACCTGCCGTCTA CCTTTGGCCAGGGCACGAAAGTTGAAATTAAACGTACGGTGGCCGCTCCCAGCGTGTTCATCTTC CCCCCCAGCGACGAGCAGCTGAAGAGTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTC TACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCA GGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCT GAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTC CAGCCCCGTGACCAAGAGCTTCAACAGGGGCG AGTGCSEQ ID NO: 128 Cys Fab-LC-E165C DIQMTQSPSSLSASVGDRVTITCRASQDISQDLA (EU)WYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAVYYCQQYYYLPSTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTCQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC SEQ ID NO: 129Cys Fab-LC-S114C DIQMTQSPSSLSASVGDRVTITCRASQDISQDLA (EU)WYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAVYYCQQYYYLPSTFGQGTKVEIKRTVAAPCVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC Anti-cKIT Ab3/Fab3/Fab′3SEQ ID NO: 1 HCDR1 (Kabat) SYAIS SEQ ID NO: 51 HCDR2 (Kabat)TIGPFEGQPRYAQKFQG SEQ ID NO: 3 HCDR3 (Kabat) GGYISDFDV SEQ ID NO: 4HCDR1 (Chothia) GGTFSSY SEQ ID NO: 52 HCDR2 (Chothia) GPFEGQSEQ ID NO: 3 HCDR3 (Chothia) GGYISDFDV SEQ ID NO: 6 HCDR1 (Combined)GGTFSSYAIS SEQ ID NO: 51 HCDR2 (Combined) TIGPFEGQPRYAQKFQG SEQ ID NO: 3HCDR3 (Combined) GGYISDFDV SEQ ID NO: 7 HCDR1 (IMGT) GGTFSSYASEQ ID NO: 53 HCDR2 (IMGT) IGPFEGQP SEQ ID NO: 9 HCDR3 (IMGT)ARGGYISDFDV SEQ ID NO: 54 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGTIGPFEGQPRYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCARGGYISDFDVWGQGTLVTVSS SEQ ID NO: 55 VH DNA CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAAAAAACCGGGCAGCAGCGTGAAAGTTAGCT GCAAAGCATCCGGAGGGACGTTTAGCAGCTATGCGATTAGCTGGGTGCGCCAGGCCCCGGGCC AGGGCCTCGAGTGGATGGGCACTATCGGTCCGTTCGAAGGCCAGCCGCGTTACGCCCAGAAATT TCAGGGCCGGGTGACCATTACCGCCGATGAAAGCACCAGCACCGCCTATATGGAACTGAGCAGC CTGCGCAGCGAAGATACGGCCGTGTATTATTGCGCGCGTGGTGGTTACATCTCTGACTTCGATG TTTGGGGCCAAGGCACCCTGGTGACTGTTAGC TCASEQ ID NO: 56 Ab HC QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGTIGPFEGQPRYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCARGGYISDFDVWGQGTLVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 57 Ab HC DNA CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAAAAAACCGGGCAGCAGCGTGAAAGTTAGCT GCAAAGCATCCGGAGGGACGTTTAGCAGCTATGCGATTAGCTGGGTGCGCCAGGCCCCGGGCC AGGGCCTCGAGTGGATGGGCACTATCGGTCCGTTCGAAGGCCAGCCGCGTTACGCCCAGAAATT TCAGGGCCGGGTGACCATTACCGCCGATGAAAGCACCAGCACCGCCTATATGGAACTGAGCAGC CTGCGCAGCGAAGATACGGCCGTGTATTATTGCGCGCGTGGTGGTTACATCTCTGACTTCGATG TTTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCAGCTAGCACCAAGGGCCCAAGTGTGTTTCC CCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGAC TACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCACACCT TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAG CTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACA AGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACT GCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGG ACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAAC AGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAAT ACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAA GGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGA ACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGA GAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAG CTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCT GCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGG CAAG SEQ ID NO: 58 Fab′ HC (EU236)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAI SWVRQAPGQGLEWMGTIGPFEGQPRYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG YISDFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL G SEQ ID NO: 59 Fab′ HC DNACAGGTGCAATTGGTGCAGAGCGGTGCCGAAGT GAAAAAACCGGGCAGCAGCGTGAAAGTTAGCTGCAAAGCATCCGGAGGGACGTTTAGCAGCTAT GCGATTAGCTGGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTGGATGGGCACTATCGGTCCG TTCGAAGGCCAGCCGCGTTACGCCCAGAAATTTCAGGGCCGGGTGACCATTACCGCCGATGAAA GCACCAGCACCGCCTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGTATTATTG CGCGCGTGGTGGTTACATCTCTGACTTCGATGTTTGGGGCCAAGGCACCCTGGTGACTGTTAGC TCAGCTAGCACCAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCG GAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAA CTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC AGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGT GAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACC CACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGA SEQ ID NO: 130 Cys Fab QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIHC (EU221)-HC- SWVRQAPGQGLEWMGTIGPFEGQPRYAQKFQG E152C (EU)RVTITADESTSTAYMELSSLRSEDTAVYYCARGG YISDFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD SEQ ID NO: 131 Fab′ HC  (EU230)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAI SWVRQAPGQGLEWMGTIGPFEGQPRYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG YISDFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP SEQ ID NO: 132 Fab′ HC(EU232)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAI SWVRQAPGQGLEWMGTIGPFEGQPRYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG YISDFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP SEQ ID NO: 133 Fab′ HC (EU236)-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAI Pro SWVRQAPGQGLEWMGTIGPFEGQPRYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG YISDFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GP SEQ ID NO: 16 LCDR1 (Kabat)RASQSISNYLA SEQ ID NO: 17 LCDR2 (Kabat) DASSLQS SEQ ID NO: 18LCDR3 (Kabat) QQYYYESIT SEQ ID NO: 19 LCDR1 (Chothia) SQSISNYSEQ ID NO: 20 LCDR2 (Chothia) DAS SEQ ID NO: 21 LCDR3 (Chothia) YYYESISEQ ID NO: 16 LCDR1 (Combined) RASQSISNYLA SEQ ID NO: 17LCDR2 (Combined) DASSLQS SEQ ID NO: 18 LCDR3 (Combined) QQYYYESITSEQ ID NO: 22 LCDR1 (IMGT) QSISNY SEQ ID NO: 20 LCDR2 (IMGT) DASSEQ ID NO: 18 LCDR3 (IMGT) QQYYYESIT SEQ ID NO: 23 VL (kappa)DIQMTQSPSSLSASVGDRVTITCRASQSISNYLA WYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYYESITFGQGT KVEIK SEQ ID NO: 24 VL DNAGATATCCAGATGACCCAGAGCCCGAGCAGCCT GAGCGCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGTCTATTTCTAACTACC TGGCTTGGTACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTAATCTACGACGCTTCTTCTCTG CAAAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCTGACCATT AGCTCTCTGCAACCGGAAGACTTTGCGACCTATTATTGCCAGCAGTACTACTACGAATCTATCACC TTTGGCCAGGGCACGAAAGTTGAAATTAAASEQ ID NO: 25 Ab/Fab′ LC (kappa) DIQMTQSPSSLSASVGDRVTITCRASQSISNYLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQYYYESITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC SEQ ID NO: 26 Ab/Fab′LC DNA GATATCCAGATGACCCAGAGCCCGAGCAGCCT GAGCGCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGTCTATTTCTAACTACC TGGCTTGGTACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTAATCTACGACGCTTCTTCTCTG CAAAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCTGACCATT AGCTCTCTGCAACCGGAAGACTTTGCGACCTATTATTGCCAGCAGTACTACTACGAATCTATCACC TTTGGCCAGGGCACGAAAGTTGAAATTAAACGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCC CCCCAGCGACGAGCAGCTGAAGAGTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTA CCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGG AGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAG CAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAG CCCCGTGACCAAGAGCTTCAACAGGGGCGAGT GCSEQ ID NO: 134 Cys Fab-LC-E165C DIQMTQSPSSLSASVGDRVTITCRASQSISNYLA (EU)WYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQYYYESITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTCQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC SEQ ID NO: 135Cys Fab-LC-S114C DIQMTQSPSSLSASVGDRVTITCRASQSISNYLA (EU)WYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQYYYESITFGQGTKVEIKRTVAAPCVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC Anti-cKIT Ab4/Fab4/Fab′4SEQ ID NO: 60 HCDR1 (Kabat) TNSAAWN SEQ ID NO: 61 HCDR2 (Kabat)RIYYRSQWLNDYAVSVKS SEQ ID NO: 62 HCDR3 (Kabat) QLTYPYTVYHKALDVSEQ ID NO: 63 HCDR1 (Chothia) GDSVSTNSA SEQ ID NO: 64 HCDR2 (Chothia)YYRSQWL SEQ ID NO: 62 HCDR3 (Chothia) QLTYPYTVYHKALDV SEQ ID NO: 65HCDR1 (Combined) GDSVSTNSAAWN SEQ ID NO: 61 HCDR2 (Combined)RIYYRSQWLNDYAVSVKS SEQ ID NO: 62 HCDR3 (Combined) QLTYPYTVYHKALDVSEQ ID NO: 66 HCDR1 (IMGT) GDSVSTNSAA SEQ ID NO: 67 HCDR2 (IMGT)IYYRSQWLN SEQ ID NO: 68 HCDR3 (IMGT) ARQLTYPYTVYHKALDV SEQ ID NO: 69 VHQVQLQQSGPGLVKPSQTLSLTCAISGDSVSTNSA AWNWIRQSPSRGLEWLGRIYYRSQWLNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARQ LTYPYTVYHKALDVWGQGTLVTVSSSEQ ID NO: 70 VH DNA CAGGTGCAATTGCAGCAGAGCGGTCCGGGCCTGGTGAAACCGAGCCAGACCCTGAGCCTGACCT GCGCGATTTCCGGAGATAGCGTGAGCACTAACTCTGCTGCTTGGAACTGGATTCGTCAGAGCCC GAGCCGTGGCCTCGAGTGGCTGGGCCGTATCTACTACCGTAGCCAGTGGCTGAACGACTATGCC GTGAGCGTGAAAAGCCGCATTACCATTAACCCGGATACTTCGAAAAACCAGTTTAGCCTGCAACT GAACAGCGTGACCCCGGAAGATACGGCCGTGTATTATTGCGCGCGTCAGCTGACTTACCCGTACA CTGTTTACCATAAAGCTCTGGATGTTTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 71 Ab HCQVQLQQSGPGLVKPSQTLSLTCAISGDSVSTNSA AWNWIRQSPSRGLEWLGRIYYRSQWLNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARQ LTYPYTVYHKALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK SEQ ID NO: 72Ab HC DNA CAGGTGCAATTGCAGCAGAGCGGTCCGGGCCTGGTGAAACCGAGCCAGACCCTGAGCCTGACCT GCGCGATTTCCGGAGATAGCGTGAGCACTAACTCTGCTGCTTGGAACTGGATTCGTCAGAGCCC GAGCCGTGGCCTCGAGTGGCTGGGCCGTATCTACTACCGTAGCCAGTGGCTGAACGACTATGCC GTGAGCGTGAAAAGCCGCATTACCATTAACCCGGATACTTCGAAAAACCAGTTTAGCCTGCAACT GAACAGCGTGACCCCGGAAGATACGGCCGTGTATTATTGCGCGCGTCAGCTGACTTACCCGTACA CTGTTTACCATAAAGCTCTGGATGTTTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGC ACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTG CCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGC TCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGC AGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCACAA GCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGC CCCCCCTGCCCAGCTCCAGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCA AGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGA GGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAA GCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAG GACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCAATCG AAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCA GCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCA GCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCC CAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTG GCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAG AAGTCCCTGAGCCTGAGCCCCGGCAAGSEQ ID NO: 73 Fab′ HC (EU236) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSTNSAAWNWIRQSPSRGLEWLGRIYYRSQWLNDYAVSV KSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARQLTYPYTVYHKALDVWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG SEQ ID NO: 74 Fab′ HC DNA CAGGTGCAATTGCAGCAGAGCGGTCCGGGCCTGGTGAAACCGAGCCAGACCCTGAGCCTGACCT GCGCGATTTCCGGAGATAGCGTGAGCACTAACTCTGCTGCTTGGAACTGGATTCGTCAGAGCCC GAGCCGTGGCCTCGAGTGGCTGGGCCGTATCTACTACCGTAGCCAGTGGCTGAACGACTATGCC GTGAGCGTGAAAAGCCGCATTACCATTAACCCGGATACTTCGAAAAACCAGTTTAGCCTGCAACT GAACAGCGTGACCCCGGAAGATACGGCCGTGTATTATTGCGCGCGTCAGCTGACTTACCCGTACA CTGTTTACCATAAAGCTCTGGATGTTTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGC ACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTG CCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGC TCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGC AGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCACAA GCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGC CCCCCCTGCCCAGCTCCAGAACTGCTGGGASEQ ID NO: 136 Cys Fab QVQLQQSGPGLVKPSQTLSLTCAISGDSVSTNSA HC (EU221)-HC-AWNWIRQSPSRGLEWLGRIYYRSQWLNDYAVSV E152C (EU)KSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARQ LTYPYTVYHKALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD SEQ ID NO: 137 Fab′ HC (EU230)QVQLQQSGPGLVKPSQTLSLTCAISGDSVSTNSA AWNWIRQSPSRGLEWLGRIYYRSQWLNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARQ LTYPYTVYHKALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CP SEQ ID NO: 138 Fab′ HC (EU232)QVQLQQSGPGLVKPSQTLSLTCAISGDSVSTNSA AWNWIRQSPSRGLEWLGRIYYRSQWLNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARQ LTYPYTVYHKALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAP SEQ ID NO: 139 Fab′ HC (EU236)-QVQLQQSGPGLVKPSQTLSLTCAISGDSVSTNSA Pro AWNWIRQSPSRGLEWLGRIYYRSQWLNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARQ LTYPYTVYHKALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGP SEQ ID NO: 75 LCDR1 (Kabat)SGDNLGDQYVS SEQ ID NO: 76 LCDR2 (Kabat) DDTDRPS SEQ ID NO: 77LCDR3 (Kabat) QSTDSKSVV SEQ ID NO: 78 LCDR1 (Chothia) DNLGDQYSEQ ID NO: 79 LCDR2 (Chothia) DDT SEQ ID NO: 80 LCDR3 (Chothia) TDSKSVSEQ ID NO: 75 LCDR1 (Combined) SGDNLGDQYVS SEQ ID NO: 76LCDR2 (Combined) DDTDRPS SEQ ID NO: 77 LCDR3 (Combined) QSTDSKSVVSEQ ID NO: 81 LCDR1 (IMGT) NLGDQY SEQ ID NO: 79 LCDR2 (IMGT) DDTSEQ ID NO: 77 LCDR3 (IMGT) QSTDSKSVV SEQ ID NO: 82 VL (lambda)DIELTQPPSVSVSPGQTASITCSGDNLGDQYVSW YQQKPGQAPVLVIYDDTDRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQSTDSKSVVFGGGTK LTVL SEQ ID NO: 83 VL DNAGATATCGAACTGACCCAGCCGCCGAGCGTGAG CGTGAGCCCGGGCCAGACCGCGAGCATTACCTGTAGCGGCGATAACCTGGGTGACCAATACGTT TCTTGGTACCAGCAGAAACCGGGCCAGGCGCCGGTGCTGGTGATCTACGACGACACTGACCGTC CGAGCGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACACCGCGACCCTGACCATTAG CGGCACCCAGGCGGAAGACGAAGCGGATTATTACTGCCAGTCTACTGACTCTAAATCTGTTGTGT TTGGCGGCGGCACGAAGTTAACCGTCCTADIELTQPPSVSVSPGQTASITCSGDNLGDQYVSW SEQ ID NO: 84 Ab/Fab′ LCYQQKPGQAPVLVIYDDTDRPSGIPERFSGSNSGN (lambda)TATLTISGTQAEDEADYYCQSTDSKSVVFGGGTK LTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNN KYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 85 Ab/Fab′ LC DNA GATATCGAACTGACCCAGCCGCCGAGCGTGAGCGTGAGCCCGGGCCAGACCGCGAGCATTACCT GTAGCGGCGATAACCTGGGTGACCAATACGTTTCTTGGTACCAGCAGAAACCGGGCCAGGCGCC GGTGCTGGTGATCTACGACGACACTGACCGTCCGAGCGGCATCCCGGAACGTTTTAGCGGATCC AACAGCGGCAACACCGCGACCCTGACCATTAGCGGCACCCAGGCGGAAGACGAAGCGGATTATT ACTGCCAGTCTACTGACTCTAAATCTGTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTAGGC CAGCCTAAGGCCGCTCCCTCCGTGACCCTGTTCCCCCCCAGCTCCGAGGAACTGCAGGCCAACA AGGCCACCCTGGTGTGCCTGATCAGCGACTTCTACCCTGGCGCCGTGACCGTGGCCTGGAAGG CCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACAACCACCCCCAGCAAGCAGAGCAACAACA AGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGAAGCTACAG CTGCCAGGTCACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC SEQ ID NO: 140 Cys Fab-LCDIELTQPPSVSVSPGQTASITCSGDNLGDQYVSW (lambda)-A143CYQQKPGQAPVLVIYDDTDRPSGIPERFSGSNSGN (EU)TATLTISGTQAEDEADYYCQSTDSKSVVFGGGTK LTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGCVTVAWKADSSPVKAGVETTTPSKQSNN KYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Anti-cKIT Ab5/Fab5/Fab′5 SEQ ID NO: 86 HCDR1 (Kabat) NYWIASEQ ID NO: 87 HCDR2 (Kabat) IIYPSNSYTLYSPSFQG SEQ ID NO: 88HCDR3 (Kabat) VPPGGSISYPAFDH SEQ ID NO: 89 HCDR1 (Chothia) GYSFTNYSEQ ID NO: 90 HCDR2 (Chothia) YPSNSY SEQ ID NO: 88 HCDR3 (Chothia)VPPGGSISYPAFDH SEQ ID NO: 91 HCDR1 (Combined) GYSFTNYWIA SEQ ID NO: 87HCDR2 (Combined) IIYPSNSYTLYSPSFQG SEQ ID NO: 88 HCDR3 (Combined)VPPGGSISYPAFDH SEQ ID NO: 92 HCDR1 (IMGT) GYSFTNYW SEQ ID NO: 93HCDR2 (IMGT) IYPSNSYT SEQ ID NO: 94 HCDR3 (IMGT) ARVPPGGSISYPAFDHSEQ ID NO: 95 VH QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIAWVRQMPGKGLEWMGIIYPSNSYTLYSPSFQGQ VTISADKSISTAYLQWSSLKASDTAMYYCARVPPGGSISYPAFDHWGQGTLVTVSS SEQ ID NO: 96 VH DNACAGGTGCAATTGGTGCAGAGCGGTGCGGAAGT GAAAAAACCGGGCGAAAGCCTGAAAATTAGCTGCAAAGGCTCCGGATATAGCTTCACTAACTACT GGATCGCTTGGGTGCGCCAGATGCCGGGCAAAGGTCTCGAGTGGATGGGCATCATCTACCCGT CTAACAGCTACACCCTGTATAGCCCGAGCTTTCAGGGCCAGGTGACCATTAGCGCGGATAAAAGC ATCAGCACCGCGTATCTGCAATGGAGCAGCCTGAAAGCGAGCGATACCGCGATGTATTATTGCG CGCGTGTTCCGCCGGGTGGTTCTATCTCTTACCCGGCTTTCGATCATTGGGGCCAAGGCACCCT GGTGACTGTTAGCTCA SEQ ID NO: 97 Ab HCQVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWI AWVRQMPGKGLEWMGIIYPSNSYTLYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVPP GGSISYPAFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID NO: 98 Ab HC DNACAGGTGCAATTGGTGCAGAGCGGTGCGGAAGT GAAAAAACCGGGCGAAAGCCTGAAAATTAGCTGCAAAGGCTCCGGATATAGCTTCACTAACTACT GGATCGCTTGGGTGCGCCAGATGCCGGGCAAAGGTCTCGAGTGGATGGGCATCATCTACCCGT CTAACAGCTACACCCTGTATAGCCCGAGCTTTCAGGGCCAGGTGACCATTAGCGCGGATAAAAGC ATCAGCACCGCGTATCTGCAATGGAGCAGCCTGAAAGCGAGCGATACCGCGATGTATTATTGCG CGCGTGTTCCGCCGGGTGGTTCTATCTCTTACCCGGCTTTCGATCATTGGGGCCAAGGCACCCT GGTGACTGTTAGCTCAGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAG TCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGA CAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAG CAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACC TATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAG CTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGGGCCTTCCGTG TTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCG TGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGG AGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTC CGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACA AGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCC AGGTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTC TGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGA ACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCT GACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGC CCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG SEQ ID NO: 99 Fab′ HC (EU236)QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWI AWVRQMPGKGLEWMGIIYPSNSYTLYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVPP GGSISYPAFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP PELLG SEQ ID NO: 100 Fab′ HC DNACAGGTGCAATTGGTGCAGAGCGGTGCGGAAGT GAAAAAACCGGGCGAAAGCCTGAAAATTAGCTGCAAAGGCTCCGGATATAGCTTCACTAACTACT GGATCGCTTGGGTGCGCCAGATGCCGGGCAAAGGTCTCGAGTGGATGGGCATCATCTACCCGT CTAACAGCTACACCCTGTATAGCCCGAGCTTTCAGGGCCAGGTGACCATTAGCGCGGATAAAAGC ATCAGCACCGCGTATCTGCAATGGAGCAGCCTGAAAGCGAGCGATACCGCGATGTATTATTGCG CGCGTGTTCCGCCGGGTGGTTCTATCTCTTACCCGGCTTTCGATCATTGGGGCCAAGGCACCCT GGTGACTGTTAGCTCAGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAG TCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGA CAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAG CAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACC TATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAG CTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGA SEQ ID NO: 141 Cys FabQVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWI HC (EU221)-HC-AWVRQMPGKGLEWMGIIYPSNSYTLYSPSFQGQ E152C (EU)VTISADKSISTAYLQWSSLKASDTAMYYCARVPP GGSISYPAFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD SEQ ID NO: 142 Fab′ HC (EU230)QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWI AWVRQMPGKGLEWMGIIYPSNSYTLYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVPP GGSISYPAFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP SEQ ID NO: 143 Fab′ HC (EU232)QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWI AWVRQMPGKGLEWMGIIYPSNSYTLYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVPP GGSISYPAFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP AP SEQ ID NO: 144 Fab′ HC (EU236)-QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWI Pro AWVRQMPGKGLEWMGIIYPSNSYTLYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVPP GGSISYPAFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGP SEQ ID NO: 101 LCDR1 (Kabat)SGDNIGSIYAS SEQ ID NO: 102 LCDR2 (Kabat) RDNKRPS SEQ ID NO: 103LCDR3 (Kabat) SVTDMEQHSV SEQ ID NO: 104 LCDR1 (Chothia) DNIGSIYSEQ ID NO: 105 LCDR2 (Chothia) RDN SEQ ID NO: 106 LCDR3 (Chothia)TDMEQHS SEQ ID NO: 101 LCDR1 (Combined) SGDNIGSIYAS SEQ ID NO: 102LCDR2 (Combined) RDNKRPS SEQ ID NO: 103 LCDR3 (Combined) SVTDMEQHSVSEQ ID NO: 107 LCDR1 (IMGT) NIGSIY SEQ ID NO: 105 LCDR2 (IMGT) RDNSEQ ID NO: 103 LCDR3 (IMGT) SVTDMEQHSV SEQ ID NO: 108 VL (lambda)DIELTQPPSVSVSPGQTASITCSGDNIGSIYASWY QQKPGQAPVLVIYRDNKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCSVTDMEQHSVFGGGT KLTVL SEQ ID NO: 109 VL DNAGATATCGAACTGACCCAGCCGCCGAGCGTGAG CGTGAGCCCGGGCCAGACCGCGAGCATTACCTGTAGCGGCGATAACATCGGTTCTATCTACGCTT CTTGGTACCAGCAGAAACCGGGCCAGGCGCCGGTGCTGGTGATCTACCGTGACAACAAACGTC CGAGCGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACACCGCGACCCTGACCATTAG CGGCACCCAGGCGGAAGACGAAGCGGATTATTACTGCTCCGTTACTGACATGGAACAGCATTCTG TGTTTGGCGGCGGCACGAAGTTAACCGTCCTADIELTQPPSVSVSPGQTASITCSGDNIGSIYASWY SEQ ID NO: 110 Ab/Fab′ LCQQKPGQAPVLVIYRDNKRPSGIPERFSGSNSGNT (lambda)ATLTISGTQAEDEADYYCSVTDMEQHSVFGGGT KLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 111 Ab/Fab′ LC DNAGATATCGAACTGACCCAGCCGCCGAGCGTGAG CGTGAGCCCGGGCCAGACCGCGAGCATTACCTGTAGCGGCGATAACATCGGTTCTATCTACGCTT CTTGGTACCAGCAGAAACCGGGCCAGGCGCCGGTGCTGGTGATCTACCGTGACAACAAACGTC CGAGCGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACACCGCGACCCTGACCATTAG CGGCACCCAGGCGGAAGACGAAGCGGATTATTACTGCTCCGTTACTGACATGGAACAGCATTCTG TGTTTGGCGGCGGCACGAAGTTAACCGTCCTAGGCCAGCCTAAGGCCGCTCCCTCCGTGACCCT GTTCCCCCCCAGCTCCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCAGCGAC TTCTACCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTG GAGACAACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGA CCCCCGAGCAGTGGAAGAGCCACAGAAGCTACAGCTGCCAGGTCACCCACGAGGGCAGCACCG TGGAGAAAACCGTGGCCCCCACCGAGTGCAGCSEQ ID NO: 145 Cys Fab-LC DIELTQPPSVSVSPGQTASITCSGDNIGSIYASWY(lambda)-A144C QQKPGQAPVLVIYRDNKRPSGIPERFSGSNSGNT (EU)ATLTISGTQAEDEADYYCSVTDMEQHSVFGGGT KLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGCVTVAWKADSSPVKAGVETTTPSKQSN NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Other anti-cKIT antibody or antibody fragment (e.g., Fab or Fab′)disclosed herein include those where the amino acids or nucleic acidsencoding the amino acids have been mutated, yet have at least 60, 70,80, 90 or 95 percent identity to the sequences described in Table 1. Insome aspects, it includes mutant amino acid sequences wherein no morethan 1, 2, 3, 4 or 5 amino acids have been mutated in the variableregions when compared with the variable regions depicted in the sequencedescribed in Table 1, while retaining substantially the same therapeuticactivity.

Since each of these antibody or antibody fragment (e.g., Fab or Fab′)can bind to cKIT, the VH, VL, heavy chain, and light chain sequences(amino acid sequences and the nucleotide sequences encoding the aminoacid sequences) can be “mixed and matched” to create other cKIT-bindingantibody or antibody fragment (e.g., Fab or Fab′). Such “mixed andmatched” cKIT-binding antibody or antibody fragment (e.g., Fab or Fab′)can be tested using the binding assays known in the art (e.g., ELISAs,and other assays described in the Example section). When these chainsare mixed and matched, a VH sequence from a particular VHNL pairingshould be replaced with a structurally similar VH sequence. Likewise aheavy chain sequence from a particular heavy chain/light chain pairingshould be replaced with a structurally similar heavy chain sequence.Likewise, a VL sequence from a particular VH/VL pairing should bereplaced with a structurally similar VL sequence. Likewise, a lightchain sequence from a particular heavy chain/light chain pairing shouldbe replaced with a structurally similar light chain sequence.

Accordingly, in one aspect, the disclosure provides for an isolatedantibody or antibody fragment (e.g., Fab or Fab′) having: a heavy chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 10, 36, 54, 69, and 95 (Table 1); and alight chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 23, 47, 82, and 108 (Table 1);wherein the antibody or antibody fragment (e.g., Fab or Fab′)specifically binds to human cKIT.

In another aspect, the disclosure provides an isolated antibody having:a heavy chain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 12, 38, 56, 71, and 97; and a light chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 25, 49, 84, and 110.

In another aspect, the disclosure provides an isolated antibody fragment(e.g., Fab′) having: a heavy chain comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 14, 40, 58, 73, and99; and a light chain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 25, 49, 84, and 110.

In another aspect, the present disclosure provides cKIT-binding antibodyor antibody fragment (e.g., Fab or Fab′) that comprises the heavy chainand light chain CDR1s, CDR2s and CDR3s as described in Table 1, orcombinations thereof. The amino acid sequences of the VH CDR1s (orHCDR1) of the antibodies or antibody fragments (e.g., Fab or Fab′) areshown in SEQ ID NOs: 1, 4, 6, 7, 27, 30, 32, 33, 60, 63, 65, 66, 86, 89,91, and 92. The amino acid sequences of the VH CDR2s (or HCDR2) of theantibodies or antibody fragments (e.g., Fab or Fab′) and are shown inSEQ ID NOs: 2, 5, 8, 28, 31, 34, 51, 52, 53, 61, 64, 67, 87, 90, and 93.The amino acid sequences of the VH CDR3s (or HCDR3) of the antibodies orantibody fragments (e.g., Fab or Fab′) are shown in SEQ ID NOs: 3, 9,29, 35, 62, 68, 88, and 94. The amino acid sequences of the VL CDR1s (orLCDR1) of the antibodies or antibody fragments (e.g., Fab or Fab′) areshown in SEQ ID NOs: 16, 19, 22, 42, 44, 46, 75, 78, 81, 101, 104, and107. The amino acid sequences of the VL CDR2s (or LCDR2) of theantibodies or antibody fragments (e.g., Fab or Fab′) are shown in SEQ IDNOs: 17, 20, 76, 79, 102, and 105. The amino acid sequences of the VLCDR3s (or LCDR3) of the antibodies or antibody fragments (e.g., Fab orFab′) are shown in SEQ ID NOs: 18, 21, 43, 45, 77, 80, 103, and 106.

Given that each of these antibodies or antibody fragments (e.g., Fab orFab′) can bind to human cKIT and that antigen-binding specificity isprovided primarily by the CDR1, 2 and 3 regions, the VH CDR1, 2 and 3sequences (or HCDR1, 2, 3) and VL CDR1, 2 and 3 sequences (or LCDR1, 2,3) can be “mixed and matched” (i.e., CDRs from different antibodies canbe mixed and match, although each antibody must contain a VH CDR1, 2 and3 and a VL CDR1, 2 and 3 to create a cKIT-binding antibody or antibodyfragment (e.g., Fab or Fab′). Such “mixed and matched” cKIT-bindingantibody or antibody fragment (e.g., Fab or Fab′) can be tested usingthe binding assays known in the art. When VH CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular VHsequence should be replaced with a structurally similar CDR sequence(s).Likewise, when VL CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular VL sequence should be replacedwith a structurally similar CDR sequence(s). It will be readily apparentto the ordinarily skilled artisan that novel VH and VL sequences can becreated by substituting one or more VH and/or VL CDR region sequenceswith structurally similar sequences from the CDR sequences shown herein.

Accordingly, the present disclosure provides an isolated antibody orantibody fragment (e.g., Fab or Fab′) comprising a heavy chain CDR1(HCDR1) comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1, 4, 6, 7, 27, 30, 32, 33, 60, 63, 65, 66,86, 89, 91, and 92; a heavy chain CDR2 (HCDR2) comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 28,31, 34, 51, 52, 53, 61, 64, 67, 87, 90, and 93; a heavy chain CDR3(HCDR3) comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 3, 9, 29, 35, 62, 68, 88, and 94; a lightchain CDR1 (LCDR1) comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 16, 19, 22, 42, 44, 46, 75, 78, 81, 101,104, and 107; a light chain CDR2 (LCDR2) comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 17, 20, 76,79, 102, and 105; and a light chain CDR3 (LCDR3) comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 18, 21,43, 45, 77, 80, 103, and 106; wherein the antibody specifically bindscKIT.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 1, a HCDR2 of SEQ ID NO: 2; a HCDR3 of SEQ ID NO: 3; a LCDR1 of SEQID NO:16; a LCDR2 of SEQ ID NO: 17; and a LCDR3 of SEQ ID NO: 18.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 4, a HCDR2 of SEQ ID NO: 5; a HCDR3 of SEQ ID NO: 3; a LCDR1 of SEQID NO:19; a LCDR2 of SEQ ID NO: 20; and a LCDR3 of SEQ ID NO: 21.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 6, a HCDR2 of SEQ ID NO: 2; a HCDR3 of SEQ ID NO: 3; a LCDR1 of SEQID NO:16; a LCDR2 of SEQ ID NO: 17; and a LCDR3 of SEQ ID NO: 18.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 7, a HCDR2 of SEQ ID NO: 8; a HCDR3 of SEQ ID NO: 9; a LCDR1 of SEQID NO: 22; a LCDR2 of SEQ ID NO: 20; and a LCDR3 of SEQ ID NO: 18.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 27, a HCDR2 of SEQ ID NO: 28; a HCDR3 of SEQ ID NO: 29; a LCDR1 ofSEQ ID NO: 42; a LCDR2 of SEQ ID NO: 17; and a LCDR3 of SEQ ID NO: 43.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 30, a HCDR2 of SEQ ID NO: 31; a HCDR3 of SEQ ID NO: 29; a LCDR1 ofSEQ ID NO: 44; a LCDR2 of SEQ ID NO: 20; and a LCDR3 of SEQ ID NO: 45.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 32, a HCDR2 of SEQ ID NO: 28; a HCDR3 of SEQ ID NO: 29; a LCDR1 ofSEQ ID NO: 42; a LCDR2 of SEQ ID NO: 17; and a LCDR3 of SEQ ID NO: 43.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 33, a HCDR2 of SEQ ID NO: 34; a HCDR3 of SEQ ID NO: 35; a LCDR1 ofSEQ ID NO: 46; a LCDR2 of SEQ ID NO: 20; and a LCDR3 of SEQ ID NO: 43.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 1, a HCDR2 of SEQ ID NO: 51; a HCDR3 of SEQ ID NO: 3; a LCDR1 of SEQID NO:16; a LCDR2 of SEQ ID NO: 17; and a LCDR3 of SEQ ID NO: 18.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 4, a HCDR2 of SEQ ID NO: 52; a HCDR3 of SEQ ID NO: 3; a LCDR1 of SEQID NO:19; a LCDR2 of SEQ ID NO: 20; and a LCDR3 of SEQ ID NO: 21.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 6, a HCDR2 of SEQ ID NO: 51; a HCDR3 of SEQ ID NO: 3; a LCDR1 of SEQID NO:16; a LCDR2 of SEQ ID NO: 17; and a LCDR3 of SEQ ID NO: 18.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 7, a HCDR2 of SEQ ID NO: 53; a HCDR3 of SEQ ID NO: 9; a LCDR1 of SEQID NO: 22; a LCDR2 of SEQ ID NO: 20; and a LCDR3 of SEQ ID NO: 18.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 60, a HCDR2 of SEQ ID NO: 61; a HCDR3 of SEQ ID NO: 62; a LCDR1 ofSEQ ID NO: 75; a LCDR2 of SEQ ID NO: 76; and a LCDR3 of SEQ ID NO: 77.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 63, a HCDR2 of SEQ ID NO: 64; a HCDR3 of SEQ ID NO: 62; a LCDR1 ofSEQ ID NO: 78; a LCDR2 of SEQ ID NO: 79; and a LCDR3 of SEQ ID NO: 80.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 65, a HCDR2 of SEQ ID NO: 61; a HCDR3 of SEQ ID NO: 62; a LCDR1 ofSEQ ID NO:75; a LCDR2 of SEQ ID NO: 76; and a LCDR3 of SEQ ID NO: 77.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 66, a HCDR2 of SEQ ID NO: 67; a HCDR3 of SEQ ID NO: 68; a LCDR1 ofSEQ ID NO: 81; a LCDR2 of SEQ ID NO: 79; and a LCDR3 of SEQ ID NO: 77.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 86, a HCDR2 of SEQ ID NO: 87; a HCDR3 of SEQ ID NO: 88; a LCDR1 ofSEQ ID NO: 101; a LCDR2 of SEQ ID NO: 102; and a LCDR3 of SEQ ID NO:103.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 89, a HCDR2 of SEQ ID NO: 90; a HCDR3 of SEQ ID NO: 88; a LCDR1 ofSEQ ID NO: 104; a LCDR2 of SEQ ID NO: 105; and a LCDR3 of SEQ ID NO:106.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 91, a HCDR2 of SEQ ID NO: 87; a HCDR3 of SEQ ID NO: 88; a LCDR1 ofSEQ ID NO: 101; a LCDR2 of SEQ ID NO: 102; and a LCDR3 of SEQ ID NO:103.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a HCDR1 of SEQ IDNO: 92, a HCDR2 of SEQ ID NO: 93; a HCDR3 of SEQ ID NO: 94; a LCDR1 ofSEQ ID NO: 107; a LCDR2 of SEQ ID NO: 105; and a LCDR3 of SEQ ID NO:103.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a heavy chainvariable region (VH) comprising the amino acid sequence of SEQ ID NO:10, and a light chain variable region (VL) comprising the amino acidsequence of SEQ ID NO: 23.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a VH comprisingthe amino acid sequence of SEQ ID NO: 36, and a VL comprising the aminoacid sequence of SEQ ID NO: 47.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a VH comprisingthe amino acid sequence of SEQ ID NO: 54, and a VL comprising the aminoacid sequence of SEQ ID NO: 23.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a VH comprisingthe amino acid sequence of SEQ ID NO: 69, and a VL comprising the aminoacid sequence of SEQ ID NO: 82.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT comprises a VH comprisingthe amino acid sequence of SEQ ID NO: 95, and a VL comprising the aminoacid sequence of SEQ ID NO: 108.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 14, and a light chain comprising theamino acid sequence of SEQ ID NO: 25.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 40, and a light chain comprising theamino acid sequence of SEQ ID NO: 49.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 58, and a light chain comprising theamino acid sequence of SEQ ID NO: 25.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 73, and a light chain comprising theamino acid sequence of SEQ ID NO: 84.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 99, and a light chain comprising theamino acid sequence of SEQ ID NO: 110.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising anamino acid sequence selected from SEQ ID NO: 119, 120 or 121, and alight chain comprising the amino acid sequence of SEQ ID NO: 25.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising anamino acid sequence selected from SEQ ID NO: 125, 126, or 127, and alight chain comprising the amino acid sequence of SEQ ID NO: 49.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising anamino acid sequence selected from SEQ ID NO: 131, 132, or 133, and alight chain comprising the amino acid sequence of SEQ ID NO: 25.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising anamino acid sequence selected from SEQ ID NO: 137, 138, or 139, and alight chain comprising the amino acid sequence of SEQ ID NO: 84.

In some embodiments, the antibody fragment (e.g., Fab′) thatspecifically binds to human cKIT comprises a heavy chain comprising anamino acid sequence selected from SEQ ID NO: 142, 143, or 144, and alight chain comprising the amino acid sequence of SEQ ID NO: 110.

In some embodiments, the antibody that specifically binds to human cKITcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:12, and a light chain comprising the amino acid sequence of SEQ ID NO:25.

In some embodiments, the antibody that specifically binds to human cKITcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:38, and a light chain comprising the amino acid sequence of SEQ ID NO:49.

In some embodiments, the antibody that specifically binds to human cKITcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:56, and a light chain comprising the amino acid sequence of SEQ ID NO:25.

In some embodiments, the antibody that specifically binds to human cKITcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:71, and a light chain comprising the amino acid sequence of SEQ ID NO:84.

In some embodiments, the antibody that specifically binds to human cKITcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:97, and a light chain comprising the amino acid sequence of SEQ ID NO:110.

In certain aspects, the antibody or antibody fragment (e.g., Fab orFab′) that specifically binds to human cKIT is an antibody or antibodyfragment (e.g., Fab or Fab′) described in Table 1.

1. Antibodies That Bind to the Same Epitope

The present disclosure provides the antibody or antibody fragment (e.g.,Fab or Fab′) that specifically binds to an epitope within theextracellular domain of the human cKIT receptor. In certain aspects theantibody or antibody fragment (e.g., Fab or Fab′) can bind to an epitopewithin domains 1-3 of the human cKIT extracellular domain.

The present disclosure also provides antibody or antibody fragment(e.g., Fab or Fab′) that binds to the same epitope as the anti-cKITantibody or antibody fragment (e.g., Fab or Fab′) described in Table 1.Additional antibody or antibody fragment (e.g., Fab or Fab′) cantherefore be identified based on their ability to cross-compete (e.g.,to competitively inhibit the binding of, in a statistically significantmanner) with other antibody or antibody fragment (e.g., Fab or Fab′) incKIT binding assays. A high throughput process for “binning” antibodiesbased upon their cross-competition is described in International PatentApplication No. WO 2003/48731. The ability of a test antibody orantibody fragment (e.g., Fab or Fab′) to inhibit the binding of antibodyor antibody fragment (e.g., Fab or Fab′) disclosed herein to a cKITprotein (e.g., human cKIT) demonstrates that the test antibody orantibody fragment (e.g., Fab or Fab′) can compete with that antibody orantibody fragment (e.g., Fab or Fab′) for binding to cKIT; such anantibody or antibody fragment (e.g., Fab or Fab′) may, according tonon-limiting theory, bind to the same or a related (e.g., a structurallysimilar or spatially proximal) epitope on the cKIT protein as theantibody or antibody fragment (e.g., Fab or Fab′) with which itcompetes. In a certain aspect, the antibody or antibody fragment (e.g.,Fab or Fab′) that binds to the same epitope on cKIT as the antibody orantibody fragment (e.g., Fab or Fab′) disclosed herein is a human orhumanized antibody or antibody fragment (e.g., Fab or Fab′). Such humanor humanized antibody or antibody fragment (e.g., Fab or Fab′) can beprepared and isolated as described herein.

2. Modification of the Framework

Antibody drug conjugates disclosed herein may comprise modifiedcKIT-binding antibody or antibody fragment (e.g., Fab or Fab′) thatcomprises modifications to framework residues within VH and/or VL, e.g.to improve the properties of the antibody drug conjugate.

In some embodiments, framework modifications are made to decreaseimmunogenicity of an antibody or antibody drug conjugate. For example,one approach is to “back-mutate” one or more framework residues to acorresponding germline sequence. Such residues can be identified bycomparing antibody framework sequences to germline sequences from whichthe antibody is derived. To “match” framework region sequences todesired germline configuration, residues can be “back-mutated” to acorresponding germline sequence by, for example, site-directedmutagenesis. Such “back-mutated” antibodies or antibody drug conjugatesare also intended to be encompassed by the invention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T-cell epitopes to thereby reduce the potentialimmunogenicity of the antibody or antibody drug conjugate. This approachis also referred to as “deimmunization” and is described in furtherdetail in U.S. Patent Publication No. 2003/0153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies can be engineered to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation. Furthermore, an antibody can be chemically modified(e.g., one or more chemical moieties can be attached to the antibody) orbe modified to alter its glycosylation, again to alter one or morefunctional properties of the antibody. Each of these aspects isdescribed in further detail below.

In one aspect, the hinge region of CH1 is modified such that the numberof cysteine residues in the hinge region is altered, e.g., increased ordecreased. This approach is described further in U.S. Pat. No. 5,677,425by Bodmer et al. The number of cysteine residues in the hinge region ofCH1 is altered to, for example, facilitate assembly of the light andheavy chains, to increase or decrease the stability of the antibody, orto allow conjugation to another molecule.

In some embodiments, the antibody or antibody fragment (e.g., Fab orFab′) disclosed herein include modified or engineered amino acidresidues, e.g., one or more cysteine residues, as sites for conjugationto a drug moiety (Junutula J R, et al.: Nat Biotechnol 2008,26:925-932). In one embodiment, the invention provides a modifiedantibody or antibody fragment (e.g., Fab or Fab′) comprising asubstitution of one or more amino acids with cysteine at the positionsdescribed herein. Sites for cysteine substitution are in the constantregions of the antibody or antibody fragment (e.g., Fab or Fab′) and arethus applicable to a variety of antibody or antibody fragment (e.g., Fabor Fab′), and the sites are selected to provide stable and homogeneousconjugates. A modified antibody or fragment can have one, two or morecysteine substitutions, and these substitutions can be used incombination with other modification and conjugation methods as describedherein. Methods for inserting cysteine at specific locations of anantibody are known in the art, see, e.g., Lyons et al, (1990) ProteinEng., 3:703-708, WO 2011/005481, WO2014/124316, WO 2015/138615. Incertain embodiments, a modified antibody comprises a substitution of oneor more amino acids with cysteine on its constant region selected frompositions 117, 119, 121,124, 139, 152, 153, 155, 157, 164, 169, 171,174,189, 191,195, 197, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292,293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, 390,392, 398, 400 and 422 of a heavy chain of the antibody, and wherein thepositions are numbered according to the EU system. In certainembodiments, a modified antibody fragment (e.g., Fab or Fab′) comprisesa substitution of one or more amino acids with cysteine on its constantregion selected from positions 121, 124, 152, 153, 155, 157, 164, 169,171, 174, 189, and 207 of a heavy chain of the antibody fragment (e.g.,Fab or Fab′), and wherein the positions are numbered according to the EUsystem. In certain embodiments, a modified antibody fragment (e.g., Fabor Fab′) comprises a substitution of one or more amino acids withcysteine on its constant region selected from positions 124, 152, 153,155, 157, 164, 174, 189, and 207 of a heavy chain of the antibodyfragment (e.g., Fab or Fab′), and wherein the positions are numberedaccording to the EU system.

In some embodiments, a modified antibody or antibody fragment (e.g., Fabor Fab′) comprises a substitution of one or more amino acids withcysteine on its constant region selected from positions 107, 108, 109,114, 126, 127, 129, 142, 143, 145, 152, 154, 156, 157, 159, 161, 165,168, 169, 170, 182, 183, 188, 197, 199, and 203 of a light chain of theantibody or antibody fragment (e.g., Fab or Fab′), wherein the positionsare numbered according to the EU system, and wherein the light chain isa human kappa light chain. In some embodiments, a modified antibody orantibody fragment (e.g., Fab or Fab′) comprises a substitution of one ormore amino acids with cysteine on its constant region selected frompositions 107, 108, 114, 126, 127, 129, 142, 159, 161, 165, 183, and 203of a light chain of the antibody or antibody fragment (e.g., Fab orFab′), wherein the positions are numbered according to the EU system,and wherein the light chain is a human kappa light chain. In someembodiments, a modified antibody or antibody fragment (e.g., Fab orFab′) comprises a substitution of one or more amino acids with cysteineon its constant region selected from positions 114, 129, 142, 145, 152,159, 161, 165, and 197 of a light chain of the antibody or antibodyfragment (e.g., Fab or Fab′), wherein the positions are numberedaccording to the EU system, and wherein the light chain is a human kappalight chain. In some embodiments, a modified antibody or antibodyfragment (e.g., Fab or Fab′) comprises a substitution of one or moreamino acids with cysteine on its constant region selected from positions107, 108, 109, 126, 143, 145, 152, 154, 156, 157, 159, 182, 183, 188,197, 199, and 203 of a light chain of the antibody or antibody fragment(e.g., Fab or Fab′), wherein the positions are numbered according to theEU system, and wherein the light chain is a human kappa light chain. Insome embodiments, a modified antibody or antibody fragment (e.g., Fab orFab′) comprises a substitution of one or more amino acids with cysteineon its constant region selected from positions 145, 152, and 197 of alight chain of the antibody or antibody fragment (e.g., Fab or Fab′),wherein the positions are numbered according to the EU system, andwherein the light chain is a human kappa light chain. In someembodiments, a modified antibody or antibody fragment (e.g., Fab orFab′) comprises a substitution of one or more amino acids with cysteineon its constant region selected from positions 114 and 165 of a lightchain of the antibody or antibody fragment (e.g., Fab or Fab′), whereinthe positions are numbered according to the EU system, and wherein thelight chain is a human kappa light chain.

In some embodiments, a modified antibody or antibody fragment (e.g., Fabor Fab′) comprises a substitution of one or more amino acids withcysteine on its constant region selected from positions 143, 145, 147,156, 159, 163, 168 of a light chain of the antibody or antibody fragment(e.g., Fab or Fab′), wherein the positions are numbered according to theEU system, and wherein the light chain is a human lambda light chain. Insome embodiments, a modified antibody or antibody fragment (e.g., Fab orFab′) comprises cysteine at position 143 (by EU numbering) of a lightchain of the antibody or antibody fragment (e.g., Fab or Fab′), whereinthe light chain is a human lambda light chain.

In certain embodiments, a modified antibody or antibody fragment (e.g.,Fab or Fab′) comprises a combination of substitution of two or moreamino acids with cysteine on its constant regions and the combination ofpositions can be selected from any of the positions listed above.

In some embodiments, a modified antibody or antibody fragment (e.g., Fabor Fab′) comprises cysteine at one or more of the following positions:position 124 of the heavy chain, position 152 of the heavy chain,position 153 of the heavy chain, position 155 of the heavy chain,position 157 of the heavy chain, position 164 of the heavy chain,position 174 of the heavy chain, position 114 of the light chain,position 129 of the light chain, position 142 of the light chain,position 159 of the light chain, position 161 of the light chain, orposition 165 of the light chain, and wherein the positions are numberedaccording to the EU system, and wherein the light chain is a kappachain. In some embodiments, a modified antibody or antibody fragment(e.g., Fab or Fab′) comprises cysteine at four of the followingpositions: position 124 of the heavy chain, position 152 of the heavychain, position 153 of the heavy chain, position 155 of the heavy chain,position 157 of the heavy chain, position 164 of the heavy chain,position 174 of the heavy chain, position 114 of the light chain,position 129 of the light chain, position 142 of the light chain,position 159 of the light chain, position 161 of the light chain, orposition 165 of the light chain, and wherein the positions are numberedaccording to the EU system, and wherein the light chain is a kappachain.

In some embodiments, a modified antibody or antibody fragment (e.g., Fabor Fab′) comprises cysteine at position 152 of the heavy chain, whereinthe position is numbered according to the EU system. In someembodiments, a modified antibody or antibody fragment (e.g., Fab orFab′) comprises cysteine at position 124 of the heavy chain, wherein theposition is numbered according to the EU system. In some embodiments, amodified antibody or antibody fragment (e.g., Fab or Fab′) comprisescysteine at position 165 of the light chain, wherein the position isnumbered according to the EU system and wherein the light chain is akappa chain. In some embodiments, a modified antibody or antibodyfragment (e.g., Fab or Fab′) comprises cysteine at position 114 of thelight chain, wherein the position is numbered according to the EU systemand wherein the light chain is a kappa chain. In some embodiments, amodified antibody or antibody fragment (e.g., Fab or Fab′) comprisescysteine at position 143 of the light chain, wherein the position isnumbered according to the EU system and wherein the light chain is alambda chain.

In some embodiments, a modified antibody or antibody fragment (e.g., Fabor Fab′) comprises cysteines at position 152 of the heavy chain andposition 165 of the light chain and wherein the positions are numberedaccording to the EU system, and wherein the light chain is a kappachain. In some embodiments, a modified antibody or antibody fragment(e.g., Fab or Fab′) comprises cysteines at position 152 of the heavychain and position 114 of the light chain and wherein the positions arenumbered according to the EU system, and wherein the light chain is akappa chain. In some embodiments, a modified antibody or antibodyfragment (e.g., Fab or Fab′) comprises cysteines at position 152 of theheavy chain and position 143 of the light chain and wherein thepositions are numbered according to the EU system, and wherein the lightchain is a lambda chain. In some embodiments, a modified antibody orantibody fragment (e.g., Fab or Fab′) comprises cysteines at position124 and position 152 of the heavy chain and wherein the positions arenumbered according to the EU system.

In some embodiments, a modified antibody or antibody fragment (e.g., Fabor Fab′) comprises cysteine at one or more of the following positions:position 155 of the heavy chain, position 189 of the heavy chain,position 207 of the heavy chain, position 145 of the light chain,position 152 of the light chain, or position 197 of the light chain, andwherein the positions are numbered according to the EU system, andwherein the light chain is a kappa chain. In some embodiments, amodified antibody or antibody fragment (e.g., Fab or Fab′) comprisescysteine at two or more (e.g., 2, 3, 4) of the following positions:position 155 of the heavy chain, position 189 of the heavy chain,position 207 of the heavy chain, position 145 of the light chain,position 152 of the light chain, or position 197 of the light chain, andwherein the positions are numbered according to the EU system, andwherein the light chain is a kappa chain.

In some embodiments, a modified antibody or antibody fragment (e.g., Fabor Fab′) comprises cysteine at one or more of the following positions:position 124 of the heavy chain, position 152 of the heavy chain,position 153 of the heavy chain, position 155 of the heavy chain,position 157 of the heavy chain, position 164 of the heavy chain,position 174 of the heavy chain, position 114 of the light chain,position 129 of the light chain, position 142 of the light chain,position 159 of the light chain, position 161 of the light chain, orposition 165 of the light chain, and wherein the positions are numberedaccording to the EU system, and wherein the light chain is a kappachain. In some embodiments, a modified antibody or antibody fragment(e.g., Fab or Fab′) comprises cysteine at two or more (e.g., 2, 3, 4) ofthe following positions: position 124 of the heavy chain, position 152of the heavy chain, position 153 of the heavy chain, position 155 of theheavy chain, position 157 of the heavy chain, position 164 of the heavychain, position 174 of the heavy chain, position 114 of the light chain,position 129 of the light chain, position 142 of the light chain,position 159 of the light chain, position 161 of the light chain, orposition 165 of the light chain, and wherein the positions are numberedaccording to the EU system, and wherein the light chain is a kappachain.

In some embodiments, a modified antibody fragment (e.g., Fab) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 118, and a light chain comprising theamino acid sequence of SEQ ID NO: 122.

In some embodiments, a modified antibody fragment (e.g., Fab) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 118, and a light chain comprising theamino acid sequence of SEQ ID NO: 123.

In some embodiments, a modified antibody fragment (e.g., Fab) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 124, and a light chain comprising theamino acid sequence of SEQ ID NO: 128.

In some embodiments, a modified antibody fragment (e.g., Fab) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 124, and a light chain comprising theamino acid sequence of SEQ ID NO: 129.

In some embodiments, a modified antibody fragment (e.g., Fab) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 130, and a light chain comprising theamino acid sequence of SEQ ID NO: 134.

In some embodiments, a modified antibody fragment (e.g., Fab) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 130, and a light chain comprising theamino acid sequence of SEQ ID NO: 135.

In some embodiments, a modified antibody fragment (e.g., Fab) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 136, and a light chain comprising theamino acid sequence of SEQ ID NO: 140.

In some embodiments, a modified antibody fragment (e.g., Fab) thatspecifically binds to human cKIT comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 141, and a light chain comprising theamino acid sequence of SEQ ID NO: 145.

3. Production of the cKIT Antibodies or Antibody Fragments

Anti-cKIT antibody or antibody fragment (e.g., Fab or Fab′) can beproduced by any means known in the art, including but not limited to,recombinant expression, chemical synthesis, or enzymatic digestion offull-length monoclonal antibodies, which can be obtained by, e.g.,hybridoma or recombinant production. Recombinant expression can be fromany appropriate host cells known in the art, for example, mammalian hostcells, bacterial host cells, yeast host cells, insect host cells, ormade by a cell-free system (e.g., Sutro's Xpress CF™ Platform,http://www.sutrobio.com/technology/).

The disclosure further provides polynucleotides encoding the antibody orantibody fragment (e.g., Fab or Fab′) described herein, e.g.,polynucleotides encoding heavy or light chain variable regions orsegments comprising the complementarity determining regions as describedherein. In some aspects, the polynucleotide encoding the heavy chainvariable regions (VH) has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with apolynucleotide selected from the group consisting of SEQ ID NOs: 11, 37,55, 70, and 96. In some aspects, the polynucleotide encoding the lightchain variable regions (VL) has at least 85%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identitywith a polynucleotide selected from the group consisting of SEQ ID NOs:24, 48, 83, and 109.

In some aspects, the polynucleotide encoding the antibody heavy chainhas at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% nucleic acid sequence identity with a polynucleotide of SEQ IDNOs: 13, 39, 57, 72, and 98. In some aspects, the polynucleotideencoding the antibody light chain has at least 85%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequenceidentity with a polynucleotide of SEQ ID NOs: 26, 50, 85, and 111.

In some aspects, the polynucleotide encoding the Fab′ heavy chain has atleast 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% nucleic acid sequence identity with a polynucleotide of SEQ ID NOs:15, 41, 59, 74, and 100. In some aspects, the polynucleotide encodingthe Fab′ light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with apolynucleotide of SEQ ID NOs: 26, 50, 85, and 111.

The polynucleotides of the present disclosure can encode only thevariable region sequence of an anti-cKIT antibody or antibody fragment(e.g., Fab or Fab′). They can also encode both a variable region and aconstant region of the antibody or antibody fragment (e.g., Fab orFab′). Some of the polynucleotide sequences encode a polypeptide thatcomprises variable regions of both the heavy chain and the light chainof one of an exemplified anti-cKIT antibody or antibody fragment (e.g.,Fab or Fab′).

The polynucleotide sequences can be produced by de novo solid-phase DNAsynthesis or by PCR mutagenesis of an existing sequence (e.g., sequencesas described in the Examples below) encoding an anti-cKIT antibody orits binding fragment. Direct chemical synthesis of nucleic acids can beaccomplished by methods known in the art, such as the phosphotriestermethod of Narang et al., Meth. Enzymol. 68:90, 1979; the phosphodiestermethod of Brown et al., Meth. Enzymol. 68:109, 1979; thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859,1981; and the solid support method of U.S. Pat. No. 4,458,066.Introducing mutations to a polynucleotide sequence by PCR can beperformed as described in, e.g., PCR Technology: Principles andApplications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press,NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Inniset al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila et al.,Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods andApplications 1:17, 1991.

Also provided in the present disclosure are expression vectors and hostcells for producing the anti-cKIT antibody or antibody fragment (e.g.,Fab or Fab′) described above. Various expression vectors can be employedto express the polynucleotides encoding the anti-cKIT antibody orantibody fragment (e.g., Fab or Fab′). Both viral-based and nonviralexpression vectors can be used to produce the antibodies in a mammalianhost cell. Nonviral vectors and systems include plasmids, episomalvectors, typically with an expression cassette for expressing a proteinor RNA, and human artificial chromosomes (see, e.g., Harrington et al.,Nat Genet. 15:345, 1997). For example, nonviral vectors useful forexpression of the anti-cKIT polynucleotides and polypeptides inmammalian (e.g., human) cells include pThioHis A, B & C, pcDNA3.1/His,pEBVHis A, B & C (Invitrogen, San Diego, Calif.), MPSV vectors, andnumerous other vectors known in the art for expressing other proteins.Useful viral vectors include vectors based on retroviruses,adenoviruses, adenoassociated viruses, herpes viruses, vectors based onSV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectorsand Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu.Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

The choice of expression vector depends on the intended host cells inwhich the vector is to be expressed. Typically, the expression vectorscontain a promoter and other regulatory sequences (e.g., enhancers) thatare operably linked to the polynucleotides encoding an anti-cKITantibody or antibody fragment (e.g., Fab or Fab′). In some aspects, aninducible promoter is employed to prevent expression of insertedsequences except under inducing conditions. Inducible promoters include,e.g., arabinose, lacZ, metallothionein promoter or a heat shockpromoter. Cultures of transformed organisms can be expanded undernoninducing conditions without biasing the population for codingsequences whose expression products are better tolerated by the hostcells. In addition to promoters, other regulatory elements may also berequired or desired for efficient expression of an anti-cKIT antibody orantibody fragment (e.g., Fab or Fab′). These elements typically includean ATG initiation codon and adjacent ribosome binding site or othersequences. In addition, the efficiency of expression may be enhanced bythe inclusion of enhancers appropriate to the cell system in use (see,e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; andBittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40enhancer or CMV enhancer may be used to increase expression in mammalianhost cells.

The expression vectors may also provide a secretion signal sequenceposition to form a fusion protein with polypeptides encoded by insertedanti-cKIT antibody or antibody fragment (e.g., Fab or Fab′) sequences.More often, the inserted anti-cKIT antibody or antibody fragment (e.g.,Fab or Fab′) sequences are linked to a signal sequences before inclusionin the vector. Vectors to be used to receive sequences encodinganti-cKIT antibody or antibody fragment (e.g., Fab or Fab′) light andheavy chain variable domains sometimes also encode constant regions orparts thereof. Such vectors allow expression of the variable regions asfusion proteins with the constant regions thereby leading to productionof intact antibodies or fragments thereof.

The host cells for harboring and expressing the anti-cKIT antibody orantibody fragment (e.g., Fab or Fab′) chains can be either prokaryoticor eukaryotic. E. coli is one prokaryotic host useful for cloning andexpressing the polynucleotides of the present disclosure. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilis, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species. In these prokaryotic hosts, one canalso make expression vectors, which typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation. Other microbes, such as yeast, can alsobe employed to express anti-cKIT antibody or antibody fragment (e.g.,Fab or Fab′) polypeptides. Insect cells in combination with baculovirusvectors can also be used.

In other aspects, mammalian host cells are used to express and producethe anti-cKIT antibody or antibody fragment (e.g., Fab or Fab′)polypeptides of the present disclosure. For example, they can be eithera hybridoma cell line expressing endogenous immunoglobulin genes (e.g.,the myeloma hybridoma clones as described in the Examples) or amammalian cell line harboring an exogenous expression vector (e.g., theSP2/0 myeloma cells exemplified below). These include any normal mortalor normal or abnormal immortal animal or human cell. For example, anumber of suitable host cell lines capable of secreting intactimmunoglobulins have been developed, including the CHO cell lines,various COS cell lines, HeLa cells, myeloma cell lines, transformedB-cells and hybridomas. The use of mammalian tissue cell culture toexpress polypeptides is discussed generally in, e.g., Winnacker, FromGenes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectorsfor mammalian host cells can include expression control sequences, suchas an origin of replication, a promoter, and an enhancer (see, e.g.,Queen et al., Immunol. Rev. 89:49-68, 1986), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences. Theseexpression vectors usually contain promoters derived from mammaliangenes or from mammalian viruses. Suitable promoters may be constitutive,cell type-specific, stage-specific, and/or modulatable or regulatable.Useful promoters include, but are not limited to, the metallothioneinpromoter, the constitutive adenovirus major late promoter, thedexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP pollllpromoter, the constitutive MPSV promoter, the tetracycline-inducible CMVpromoter (such as the human immediate-early CMV promoter), theconstitutive CMV promoter, and promoter-enhancer combinations known inthe art.

Methods for introducing expression vectors containing the polynucleotidesequences of interest vary depending on the type of cellular host. Forexample, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other cellular hosts (see generallySambrook et al., supra). Other methods include, e.g., electroporation,calcium phosphate treatment, liposome-mediated transformation, injectionand microinjection, ballistic methods, virosomes, immunoliposomes,polycation:nucleic acid conjugates, naked DNA, artificial virions,fusion to the herpes virus structural protein VP22 (Elliot and O'Hare,Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivotransduction. For long-term, high-yield production of recombinantproteins, stable expression will often be desired. For example, celllines which stably express anti-cKIT antibody or antibody fragment(e.g., Fab or Fab′) chains can be prepared using expression vectorswhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following introduction of thevector, cells may be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth of cells which successfully express the introducedsequences in selective media. Resistant, stably transfected cells can beproliferated using tissue culture techniques appropriate to the celltype.

Antibody fragments, such as Fab or Fab′ may be produced by proteolyticcleavage of immunoglobulin molecules, using enzymes such as papain (toproduce Fab fragments), or pepsin (to produce Fab′ fragments), etc.Compared to Fab fragments, Fab′ fragments also contain the hinge regionwhich includes the two natural cysteines that form disulfide bondsbetween two heavy chains of an immunoglobulin molecule.

Therapeutic Uses

The conjugates of the present disclosure are useful in a variety ofapplications including, but not limited to, for ablating hematopoieticstem cells in a patient in need thereof, e.g., a hematopoietic stem celltransplantation recipient. Accordingly, provided herein are methods ofablating hematopoietic stem cells in a patient in need thereof byadministering to the patient an effective amount of any of theconjugates described herein. Provided herein are also methods ofconditioning a hematopoietic stem cell transplantation patient (e.g., atransplant recipient) by administering to the patient an effectiveamount of any of the conjugates described herein, and allowing asufficient period of time for the conjugates to clear from the patient'scirculation before performing hematopoietic stem cell transplantation tothe patient. The conjugates can be administered to the patientintravenously. Also provided are use of any of the conjugates orpharmaceutical compositions described herein for ablating hematopoieticstem cells in a patient in need thereof. Further provided are use of anyof the conjugates or pharmaceutical compositions described herein in themanufacture of a medicament for ablating hematopoietic stem cells in apatient in need thereof.

Endogenous hematopoietic stem cells usually reside within bone marrowsinusoids. This physical environment in which stem cells reside isreferred to as the stem cell microenvironment, or stem cell niche. Thestromal and other cells involved in this niche provide soluble and boundfactors, which have a multitude of effects. Various models have beenproposed for the interaction between hematopoietic stem cells and theirniche. For example, a model has been suggested where, when a stem celldivides, only one daughter remains in the niche and the other daughtercell leaves the niche to differentiate. It has been proposed that theefficiency of engraftment can be enhanced by selective depletion ofendogenous hematopoietic stem cells, thereby opening the stem cellniches for the engraftment of donor stem cells (see e.g., WO2008/067115).

Hematopoietic stem cell (HSC) transplantation, or bone marrowtransplantation (as called earlier), is an established treatment for awide range of diseases that affect the body's blood stem cells such asleukemia, severe anemia, immune defects, and some enzyme deficiencydiseases. These illnesses often lead to the patient needing to have hisbone marrow replaced by new, healthy blood cells.

HSC transplantation is often allogeneic, which means that the patientreceives stem cells from another individual of the same species, eithera sibling, matched related, haploidentical related or unrelated,volunteer donor. It is estimated that about 30% of patients in need ofhematopoietic stem cell transplantation have access to a sibling whosetissue type is suitable. The other 70% of patients must rely on thematching of an unrelated, volunteer donor or the availability of ahaploidentical, related donor. It is important that the characteristicsof donor and patient cells are comparable. The hematopoietic stem celltransplantation could also be autologous, in which the transplantedcells are originating from the subject itself, i.e., the donor and therecipient are the same individual. Further, the transplantations couldbe syngeneic, i.e., from a genetically identical individual such as atwin. In an additional aspect the transplantations could be xenogeneic,i.e., originating from a different species, which is of interest whenthere are not sufficient donors of the same species, such as for organtransplantations.

Before the HSC transplantation, patients usually undergo a pre-treatmentor conditioning method. The purpose of this pre-treatment orconditioning is to remove as many undesired cells (e.g.,malignant/cancer cells) in the body as possible, to minimize rejection,and/or to open up stem cell niches by depletion of endogenous HSCs forefficient engraftment of donor stem cells into those niches. Donor'shealthy HSCs are then given to the patient intravenously, or in somecases intraosseously. Many risks, however, are associated with HSCtransplantation, including poor engraftment, immunological rejection,graft-versus-host disease (GVHD), or infection. Although the donor andthe patient's cells appear to be equal in terms of tissue type, e.g.,the MHC molecules are matched (or haploidentical); there are still minordifferences between these individuals that immune cells can perceive asdangerous. This means that the new immune system (white blood cells fromthe new stem cells) perceive the new body as “foreign”, which provokesan immune attack. This reaction, called graft-versus-host disease(GVHD), can become life-threatening to the patient. Patients after HSCtransplantation also have an increased risk of infections due to absenceof white blood cells before the new marrow begins to function. Thisperiod can in some cases last for many months until the new immunesystem have matured. Some of these opportunistic infections may belife-threatening.

Thus, there is a need for improving the conditioning and transplantationmethods and decreasing the risks associated with HSC transplantation andincreasing its effectiveness for various disorders. Provided herein arenew antibody drug conjugates that, by specifically killing therecipient's endogenous HSCs prior to transplantation but not all otherimmune cells, keep a partially active immune defense to combatinfections right after transplantation, but at the same time provide anindirect immunosuppressive effect due to the subject's inability to formnew immune cells from its own HSCs. Since the pre-treatment can bemilder than chemotherapy or radiation, and with less serious sideeffects, it might induce less GVHD in transplant patients.

The antibody drug conjugates described herein could be used to ablateendogenous hematopoietic stem cell, e.g., in apre-treatment/conditioning method before hematopoietic stem celltransplantation. For example, the conjugates of the invention could beused to treat any non-malignant condition/disorder wherein stem celltransplantation could be beneficial, such as Severe aplastic anemia(SAA), Wiskott Aldrich Syndrome, Hurlers Syndrome, familialhaemophagocytic lymphohistiocytosis (FHL), Chronic granulomatous disease(CGD), Kostmanns syndrome, Severe immunodeficiency syndrome (SCID),other autoimmune disorders such as SLE, Multiple sclerosis, IBD, CrohnsDisease, Ulcerative colitis, Sjogrens syndrome, vasculitis, Lupus,Myasthenia Gravis, Wegeners disease, inborn errors of metabolism and/orother immunodeficiencies.

Further, the conjugates of the invention could be used to treat anymalignant condition/disorder wherein stem cell transplantation could bebeneficial, such as hematologic diseases, hematological malignancies orsolid tumors (e.g., renal cancer, hepatic cancer, pancreatic cancer).Common types of hematological diseases/malignancies that could betreated with the claimed methods and antibodies are leukemias, lymphomasand myelodysplastic syndromes. Leukemia is a type of cancer of the bloodor bone marrow characterized by an abnormal increase of immature whiteblood cells called blast cells, and the term leukemia includes: acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), acutemonocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), chronicmyelogenous leukemia (CML) and other leukemias such as hairy cellleukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granularlymphocytic leukemia and adult T-cell leukemia. In one aspect of theinvention, the leukemia treated is acute leukemia. In a further aspect,the leukemia is ALL, AML or AMoL. Lymphomas include precursor T-cellleukemia/lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse largeB cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocyticleukemia/lymphoma, MALT lymphoma, Mycosis fungoides, Peripheral T-celllymphoma not otherwise specified, Nodular sclerosis form of Hodgkinlymphoma Mixed-cellularity subtype of Hodgkin lymphoma. Myelodysplasticsyndrome (MDS) is the name of a group of conditions that occur when theblood-forming cells in the bone marrow are damaged. This damage leads tolow numbers of one or more type of blood cells. MDS is subdivided into 7categories; Refractory cytopenia with unilineage dysplasia (RCUD),Refractory anemia with ringed sideroblasts (RARS), Refractory cytopeniawith multilineage dysplasia (RCMD), Refractory anemia with excessblasts-1 (RAEB-1), Refractory anemia with excess blasts-2 (RAEB-2),Myelodysplastic syndrome, unclassified (MDS-U), and Myelodysplasticsyndrome associated with isolated del (5q).

In some embodiments, a patient in need of ablating hematopoietic stemcells (e.g., a hematopoietic stem cell transplantation recipient) mayhave an inherited immunodeficient disease, an autoimmune disorder, ahematopoietic disorder, or inborn errors of metabolism.

In some embodiments, the hematopoietic disorder can be selected from anyof the following: Acute myeloid leukemia (AML), Acute lymphoblasticleukemia (ALL), acute monocytic leukemia (AMoL), Chronic myeloidleukemia (CML), Chronic lymphocytic leukemia (CLL), Myeloproliferativedisorders, Myelodysplastic syndromes, Multiple myeloma, Non-Hodgkinlymphoma, Hodgkin disease, Aplastic anemia, Pure red cell aplasia,Paroxysmal nocturnal hemoglobinuria, Fanconi anemi, Thalassemia major,Sickle cell anemia, Severe combined immunodeficiency, Wiskott-Aldrichsyndrome, Hemophagocytic lymphohistiocytosis.

Inborn errors of metabolism are also known as inherited metabolicdiseases (IMB) or congenital metabolic diseases, which are a class ofgenetic diseases that include congenital disorders of carbohydratemetabolism, amino acid metabolism, organic acid metabolism, or lysosomalstorage diseases. In some embodiments, inborn errors of metabolism areselected from mucopolysaccharidosis, Gaucher disease, metachromaticleukodystrophies, or adrenoleukodystrophies.

Further, the conjugates of the invention could be used to treat agastrointestinal stromal tumor (GIST), such as GIST that is cKITpositive. In some embodiments, the conjugates of the invention could beused to treat GIST that expresses wild-type cKIT. In some embodiments,the conjugates of the invention could be used to treat GIST that isresistant to a treatment, e.g., imatinib (Glivec®/Gleevec®).

Combination Therapy

In certain instances, an antibody drug conjugate of the presentdisclosure can be used in combination with another conditioning regimentsuch as radiation therapy or chemotherapy.

In certain instances, an antibody drug conjugate of the presentdisclosure can be used in combination with another therapeutic agent,such as an anti-cancer agent, anti-nausea agent (or anti-emetic), painreliever, mobilizing agent, or combinations thereof.

General chemotherapeutic agents considered for use in combinationtherapies include anastrozole (Arimidex®), bicalutamide (Casodex®),bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection(Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclosporine (Sandimmune®, Neoral® orRestasis®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosinearabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®),dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan),daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposomeinjection (DaunoXome®), dexamethasone, docetaxel (Taxotere®),doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®),fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®),flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxycitidine),hydroxyurea (Hydrea®), Idarubicin (Idamycin®), ifosfamide (IFEX®),irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium,melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate(Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®),phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 withcarmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide(Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecanhydrochloride for injection (Hycamptin®), vinblastine (Velban®),vincristine (Oncovin®), and vinorelbine (Navelbine®).

In some embodiments, the antibody drug conjugate of the presentdisclosure can be used in combination with a CD47 blocker, e.g., ananti-CD47 antibody or fragment thereof. It was reported that ananti-CD47 microbody that blocks the interaction between CD47 and signalregulatory protein alpha (SIRPa) can enhance depletion of endogenousHSCs by a naked anti-c-Kit antibody (Chhabra et al., ScienceTranslational Medicine 8 (351), 351 ra105).

In some embodiments, the antibody drug conjugate of the presentdisclosure can be used in combination with another antibody or fragmentthereof that specifically binds to hematopoietic stem cells orhematopoietic progenitor cells, e.g., anti-CD45 antibody or fragmentthereof, anti-CD34 antibody or fragment thereof, anti-CD133 antibody orfragment thereof, anti-CD59 antibody or fragment thereof, or anti-CD90antibody or fragment thereof. In some embodiments, the antibody drugconjugates of the present disclosure can be used in combination with aDyrkla inhibitor, such as Harmine, INDY, ML 315 hydrochloride, ProlNDY,Tocris™ TC-S 7044, Tocris™ TG 003, FINDY, TBB, DMAT, CaNDY, etc.

In some embodiments, the antibody drug conjugate of the presentdisclosure can be used in combination with one or more immunesuppressors, such as glucocorticoids, e.g., prednisone, dexamethasone,and hydrocortisone; cytostatics, e.g., alkylating agents,antimetabolites, methotrexate, azathioprine, mercaptopurine,dactinomycin, etc.; drugs acting on immunophilins, e.g., tacrolimus(Prograf®, Astograf XL® or Envarsus XR®), sirolimus (rapamycin orRapamune®) and everolimus; interferons; opoids; TNF binding proteins;mycophenolate; fingolimod; myriocin; etc. In some embodiments, theantibody drug conjugate of the present disclosure can be used incombination with one or more agents that specifically deplete T cells,such as Fludarabine, Ciclosporin, anti-CD52 antibody, e.g., Alemtuzumab,Antithymocyte globulin (ATG), anti-CD3 antibody or fragment thereof,anti-CD4 antibody or fragment thereof, anti-CD8 antibody or fragmentthereof, or anti-human TCR a/1 antibody or fragment thereof. T celldepletion therapies can reduce host versus graft reaction, which couldlead to rejection of a transplant.

In some embodiments, the antibody drug conjugate of the presentdisclosure can be used in combination with one or more agents selectedfrom plerixafor (also known as AMD3100, Mozobil®),granulocyte-macrophage colony stimulating factor (GM-CSF), e.g.,sargramostim (Leukine®), or granulocyte-colony stimulating factor(G-CSF), e.g., filgrastim or pegfilgrastim (Zarzio®, Zarxio®, Neupogen®,Neulasta®, Nufil®, Religrast®, Emgrast®, Neukine®, Grafeel®, Imumax®,Filcad®).

In one aspect, an antibody drug conjugate of the present disclosure iscombined in a pharmaceutical combination formulation, or dosing regimenas combination therapy, with a second compound having anti-cancerproperties. The second compound of the pharmaceutical combinationformulation or dosing regimen can have complementary activities to theconjugate of the combination such that they do not adversely affect eachother.

The term “pharmaceutical combination” as used herein refers to either afixed combination in one dosage unit form, or non-fixed combination or akit of parts for the combined administration where two or moretherapeutic agents may be administered independently at the same time orseparately within time intervals, especially where these time intervalsallow that the combination partners show a cooperative, e.g. synergisticeffect.

The term “combination therapy” refers to the administration of two ormore therapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients. Alternatively, such administration encompassesco-administration in multiple, or in separate containers (e.g.,capsules, powders, and liquids) for each active ingredient. Powdersand/or liquids may be reconstituted or diluted to a desired dose priorto administration. In addition, such administration also encompasses useof each type of therapeutic agent in a sequential manner, either atapproximately the same time or at different times. In either case, thetreatment regimen will provide beneficial effects of the drugcombination in treating the conditions or disorders described herein.

The combination therapy can provide “synergy” and prove “synergistic”,i.e., the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect can be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect can be attained when the compounds are administered or deliveredsequentially, e.g., by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e., serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

Pharmaceutical Compositions

To prepare pharmaceutical or sterile compositions including one or moreantibody drug conjugates described herein, the provided conjugate(s) canbe mixed with a pharmaceutically acceptable carrier or excipient.

Formulations of therapeutic and diagnostic agents can be prepared bymixing with physiologically acceptable carriers, excipients, orstabilizers in the form of, e.g., lyophilized powders, slurries, aqueoussolutions, lotions, or suspensions (see, e.g., Hardman et al., Goodmanand Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, NewYork, N.Y., 2001; Gennaro, Remington: The Science and Practice ofPharmacy, Lippincott, Williams, and Wilkins, New York, N.Y., 2000; Avis,et al. (eds.), Pharmaceutical Dosage Forms: Parenteral Medications,Marcel Dekker, N Y, 1993; Lieberman, et al. (eds.), PharmaceuticalDosage Forms: Tablets, Marcel Dekker, N Y, 1990; Lieberman, et al.(eds.) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY, 1990; Weiner and Kotkoskie, Excipient Toxicity and Safety, MarcelDekker, Inc., New York, N.Y., 2000).

In some embodiments, the pharmaceutical composition comprising theantibody conjugate of the present invention is a lyophilisatepreparation. In certain embodiments a pharmaceutical compositioncomprising the antibody conjugate is a lyophilisate in a vial containingan antibody conjugate, histidine, sucrose, and polysorbate 20. Incertain embodiments the pharmaceutical composition comprising theantibody conjugate is a lyophilisate in a vial containing an antibodyconjugate, sodium succinate, and polysorbate 20. In certain embodimentsthe pharmaceutical composition comprising the antibody conjugate is alyophilisate in a vial containing an antibody conjugate, trehalose,citrate, and polysorbate 8. The lyophilisate can be reconstituted, e.g.,with water, saline, for injection. In a specific embodiment, thesolution comprises the antibody conjugate, histidine, sucrose, andpolysorbate 20 at a pH of about 5.0. In another specific embodiment thesolution comprises the antibody conjugate, sodium succinate, andpolysorbate 20. In another specific embodiment, the solution comprisesthe antibody conjugate, trehalose dehydrate, citrate dehydrate, citricacid, and polysorbate 8 at a pH of about 6.6. For intravenousadministration, the obtained solution will usually be further dilutedinto a carrier solution.

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. In certainembodiments, an administration regimen maximizes the amount oftherapeutic delivered to the patient consistent with an acceptable levelof side effects. Accordingly, the amount of biologic delivered dependsin part on the particular entity and the severity of the condition beingtreated. Guidance in selecting appropriate doses of antibodies,cytokines, and small molecules are available (see, e.g., Wawrzynczak,Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, U K, 1996;Kresina (ed.), Monoclonal Antibodies, Cytokines and Arthritis, MarcelDekker, New York, N.Y., 1991; Bach (ed.), Monoclonal Antibodies andPeptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.,1993; Baert et al., New Engl. J. Med. 348:601-608, 2003; Milgrom et al.,New Engl. J. Med. 341:1966-1973, 1999; Slamon et al., New Engl. J. Med.344:783-792, 2001; Beniaminovitz et al., New Engl. J. Med. 342:613-619,2000; Ghosh et al., New Engl. J. Med. 348:24-32, 2003; Lipsky et al.,New Engl. J. Med. 343:1594-1602, 2000).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors known in themedical arts.

Compositions comprising the antibody conjugate of the invention can beprovided by continuous infusion, or by doses at intervals of, e.g., oneday, one week, or 1-7 times per week, once every other week, once everythree weeks, once every four weeks, once every five weeks, once everysix weeks, once every seven weeks, or once every eight weeks. Doses maybe provided intravenously, subcutaneously, or intraosseously. A specificdose protocol is one involving the maximal dose or dose frequency thatavoids significant undesirable side effects.

For the antibody conjugates of the invention, the dosage administered toa patient may be 0.0001 mg/kg to 100 mg/kg of the patient's body weight.The dosage may be between 0.001 mg/kg and 50 mg/kg, 0.005 mg/kg and 20mg/kg, 0.01 mg/kg and 20 mg/kg, 0.02 mg/kg and 10 mg/kg, 0.05 and 5mg/kg, 0.1 mg/kg and 10 mg/kg, 0.1 mg/kg and 8 mg/kg, 0.1 mg/kg and 5mg/kg, 0.1 mg/kg and 2 mg/kg, 0.1 mg/kg and 1 mg/kg of the patient'sbody weight. The dosage of the antibody conjugate may be calculatedusing the patient's weight in kilograms (kg) multiplied by the dose tobe administered in mg/kg.

Doses of the antibody conjugates the invention may be repeated and theadministrations may be separated by less than 1 day, at least 1 day, 2days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75days, 3 months, 4 months, 5 months, or at least 6 months. In someembodiments, an antibody conjugate of the invention is administeredtwice weekly, once weekly, once every two weeks, once every three weeks,once every four weeks, or less frequently.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the method, route and dose of administration and the severityof side effects (see, e.g., Maynard et al., A Handbook of SOPs for GoodClinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, GoodLaboratory and Good Clinical Practice, Urch Publ., London, U K, 2001).

The route of administration may be by, e.g., topical or cutaneousapplication, injection or infusion by subcutaneous, intravenous,intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, intracerebrospinal, intralesional administration, or bysustained release systems or an implant (see, e.g., Sidman et al.,Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res.15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982; Epstein et al.,Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985; Hwang et al., Proc. Natl.Acad. Sci. USA 77:4030-4034, 1980; U.S. Pat. Nos. 6,350,466 and6,316,024). Where necessary, the composition may also include asolubilizing agent or a local anesthetic such as lidocaine to ease painat the site of the injection, or both. In addition, pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064,5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, eachof which is incorporated herein by reference their entirety.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, orradiation (such as total body irradiation (TBI)), are known in the art(see, e.g., Hardman et al., (eds.) (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, 10.sup.th ed., McGraw-Hill, NewYork, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics forAdvanced Practice:A Practical Approach, Lippincott, Williams & Wilkins,Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy andBiotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effectiveamount of therapeutic may decrease the symptoms by at least 10%; by atleast 20%; at least about 30%; at least 40%, or at least 50%.

Additional therapies, which can be administered in combination with theantibody conjugates of the invention may be administered less than 5minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hourapart, at about 1 to about 2 hours apart, at about 2 hours to about 3hours apart, at about 3 hours to about 4 hours apart, at about 4 hoursto about 5 hours apart, at about 5 hours to about 6 hours apart, atabout 6 hours to about 7 hours apart, at about 7 hours to about 8 hoursapart, at about 8 hours to about 9 hours apart, at about 9 hours toabout 10 hours apart, at about 10 hours to about 11 hours apart, atabout 11 hours to about 12 hours apart, at about 12 hours to 18 hoursapart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hoursto 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hoursapart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hoursto 96 hours apart, or 96 hours to 120 hours apart from the antibodyconjugates of the invention. The two or more therapies may beadministered within one same patient visit.

The invention provides protocols for the administration ofpharmaceutical composition comprising antibody conjugates of theinvention alone or in combination with other therapies to a subject inneed thereof. The therapies of the combination therapies of the presentinvention can be administered concomitantly or sequentially to asubject. The therapy of the combination therapies of the presentinvention can also be cyclically administered. Cycling therapy involvesthe administration of a first therapy for a period of time, followed bythe administration of a second therapy for a period of time andrepeating this sequential administration, i.e., the cycle, in order toreduce the development of resistance to one of the therapies (e.g.,agents) to avoid or reduce the side effects of one of the therapies(e.g., agents), and/or to improve, the efficacy of the therapies.

The therapies of the combination therapies of the invention can beadministered to a subject concurrently.

The term “concurrently” is not limited to the administration oftherapies at exactly the same time, but rather it is meant that apharmaceutical composition comprising antibodies or fragments thereofthe invention are administered to a subject in a sequence and within atime interval such that the antibodies or antibody conjugates of theinvention can act together with the other therapy(ies) to provide anincreased benefit than if they were administered otherwise. For example,each therapy may be administered to a subject at the same time orsequentially in any order at different points in time; however, if notadministered at the same time, they should be administered sufficientlyclose in time so as to provide the desired therapeutic effect. Eachtherapy can be administered to a subject separately, in any appropriateform and by any suitable route. In various embodiments, the therapiesare administered to a subject less than 5 minutes apart, less than 15minutes apart, less than 30 minutes apart, less than 1 hour apart, atabout 1 hour apart, at about 1 hour to about 2 hours apart, at about 2hours to about 3 hours apart, at about 3 hours to about 4 hours apart,at about 4 hours to about 5 hours apart, at about 5 hours to about 6hours apart, at about 6 hours to about 7 hours apart, at about 7 hoursto about 8 hours apart, at about 8 hours to about 9 hours apart, atabout 9 hours to about 10 hours apart, at about 10 hours to about 11hours apart, at about 11 hours to about 12 hours apart, 24 hours apart,48 hours apart, 72 hours apart, or 1 week apart. In other embodiments,two or more therapies are administered within the same patient visit.

The combination therapies can be administered to a subject in the samepharmaceutical composition. Alternatively, the therapeutic agents of thecombination therapies can be administered concurrently to a subject inseparate pharmaceutical compositions. The therapeutic agents may beadministered to a subject by the same or different routes ofadministration.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

Examples Example 1: Generation of Anti-cKIT ADC

Preparation of Anti-cKit Antibodies and Antibody Fragments with orwithout Site-Specific Cysteine Mutations

Human anti-cKIT antibodies and antibody fragments were generated asdescribed previously in WO2014150937 and WO2016020791.

DNA encoding variable regions of the heavy and light chains of ananti-cKit antibody were amplified from a vector isolated in a phagedisplay based screen and cloned into mammalian expression vectors thatcontain the constant regions of human IgG1 heavy chain and human kappalight chain or lambda light chain. Vectors contain a CMV promoter and asignal peptide (MPLLLLLPLLWAGALA (SEQ ID NO: 151) for heavy chain andMSVLTQVLALLLLWLTGTRC (SEQ ID NO: 152) for light chain, and appropriatesignal and selection sequences for amplification of DNA in a bacterialhost, e.g. E. coli DH5alpha cells, transient expression in mammaliancells, e.g. HEK293 cells, or stable transfection into mammalian cells,e.g. CHO cells. To introduce Cys mutations, site-directed mutagenesisPCR was conducted with oligos designed to substitute single Cys residuesat certain site in the constant regions of the heavy chain or lightchain coding sequences. Examples of Cys substitution mutations are E152Cor S375C of heavy chain; E165C or S114C of kappa light chain; or A143Cof the lambda light chain (all EU numbering). In some cases, two or moreCys mutations were combined to make an antibody with multiple Cyssubstitutions, for example HC-E152C-S375C, lambda LC-A143C-HC-E152C,kappa LC-E165C-HC-E152C, or kappa LC-S114C-HC-E152C (all EU numbering).To generate plasmids encoding antibody fragments, mutagenesis PCR wasconducted with oligos designed to remove or modify a portion of theheavy chain constant region. For example, a PCR was performed to removeresidues 222-447 (EU numbering) of the heavy chain constant region suchthat a stop codon was encoded directly after residue 221 (EU number) inorder to make an expression construct for a Fab fragment. For example, aPCR was performed to remove residues 233-447 (EU numbering) of the heavychain constant region such that a stop codon was encoded directly afterresidue 232 (EU number) in order to make an expression construct for aFab′ fragment including the two Cys residues of the IgG1 hinge.

Anti-cKit antibodies, antibody fragments, and Cys mutant antibodies orantibody fragments were expressed in 293 Freestyle™ cells byco-transfecting heavy chain and light chain plasmids using transienttransfection methods as described previously (Meissner, et al.,Biotechnol Bioeng. 75:197-203 (2001)). The expressed antibodies werepurified from the cell supernatants by standard affinity chromatographymethods using an appropriate resin such as Protein A, Protein G, Capto-Lor LambdaFabSelect resins. Alternatively, anti-cKit antibodies, antibodyfragments, and Cys mutant antibodies or antibody fragments wereexpressed in a CHO by co-transfecting a heavy chain vector and a lightchain vector into CHO cells. Cells underwent selection, and stablytransfected cells were then cultured under conditions optimized forantibody production. Antibodies were purified from the cell supernatantsas above.

Reduction, Re-Oxidation and Coniugation of Anti-cKit Antibodies andAntibody Fragments to Toxins

Compounds comprised of a reactive moiety, e.g. a maleimide group, forreaction to a thiol group (Cys side chain) on the antibody or antibodyfragment, a linker as described, and a functional moiety, such as anauristatin or other toxin, were conjugated to Cys residues, native orengineered into the antibody using methods described previously (e.g.,in WO2014124316, WO2015138615, Junutula J R, et al., NatureBiotechnology 26:925-932 (2008)).

Because engineered Cys residues in antibodies expressed in mammaliancells are modified by adducts (disulfides) such as glutathione (GSH)and/or cysteine during biosynthesis (Chen et al. 2009), the modified Cysas initially expressed is unreactive to thiol reactive reagents such asmaleimido or bromo-acetamide or iodo-acetamide groups. To conjugateengineered Cys residues, glutathione or cysteine adducts need to beremoved by reducing disulfides, which generally entails reducing alldisulfides in the expressed antibody. Because native Cys residues inantibodies and antibody fragments generally form disulfide bonds toother Cys residues in the antibody or antibody fragment, these are alsounreactive to thiol reactive reagents until the disulfides are reduced.Reduction of disulfides can be accomplished by first exposing antibodyto a reducing agent such as dithiothreitol (DTT), cysteine, orTris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl). Optionally, thereducing agent can be removed to allow re-oxidation of all nativedisulfide bonds of the antibody or antibody fragment to restore and/orstabilize the functional antibody structure.

In cases where an antibody or antibody fragment was conjugated only atengineered Cys residues, in order to reduce native disulfide bonds anddisulfide bond between the cysteine or GSH adducts of engineered Cysresidue(s), freshly prepared DTT was added to purified Cys mutantantibodies, to a final concentration of 10 mM or 20 mM. After antibodyincubation with DTT at 37° C. for 1 hour, mixtures were dialyzed againstPBS for three days with daily buffer exchange to remove DTT andre-oxidize native disulfide bonds. The re-oxidation process wasmonitored by reverse-phase HPLC, which is able to separate antibodytetramer from individual heavy and light chain molecules. Reactions wereanalyzed on a PRLP-S 4000A column (50 mm×2.1 mm, Agilent) heated to 800°C. and column elution was carried out by a linear gradient of 30-60%acetonitrile in water containing 0.1% TFA at a flow rate of 1.5 ml/min.The elution of proteins from the column was monitored at 280 nm.Dialysis was allowed to continue until reoxidation was complete.Reoxidation restores intra-chain and interchain disulfides, whiledialysis allows cysteines and glutathiones connected to thenewly-introduced Cys residue(s) to dialyze away. After re-oxidation,maleimide-containing compounds were added to re-oxidized antibodies orantibody fragments in PBS buffer (pH 7.2) at ratios of typically 1.5:1,2:1, or 5:1 to engineered Cys, and incubations were carried out for 1hour. Typically, excess free compound was removed by purification overProtein A or other appropriate resin by standard methods followed bybuffer exchange into PBS.

Alternatively, antibodies or antibody fragments with engineered Cyssites were reduced and re-oxidized using an on-resin method. Protein ASepharose beads (1 ml per 10 mg antibody) were equilibrated in PBS (nocalcium or magnesium salts) and then added to an antibody sample inbatch mode. A stock of 0.5 M cysteine was prepared by dissolving 850 mgof cysteine HCl in 10 ml of a solution prepared by adding 3.4 g of NaOHto 250 ml of 0.5 M sodium phosphate pH 8.0 and then 20 mM cysteine wasadded to the antibody/bead slurry, and mixed gently at room temperaturefor 30-60 minutes. Beads were loaded to a gravity column and washed with50 bed volumes of PBS in less than 30 minutes. Then the column wascapped with beads resuspended in one bed volume of PBS. To modulate therate of re-oxidation, 50 nM to 1 pIM copper chloride was optionallyadded. The re-oxidation progress was monitored by removing a small testsample of the resin, eluting in IgG Elution buffer (Thermo), andanalyzing by RP-HPLC as described above. Once re-oxidation progressed todesired completeness, conjugation could be initiated immediately byaddition of 2-3 molar excess of compound over engineered cysteines, andallowing the mixture to react for 5-10 minutes at room temperaturebefore the column was washed with at least 20 column volumes of PBS.Antibody conjugates were eluted with IgG elution buffer and neutralizedwith 0.1 volumes 0.5 M sodium phosphate pH 8.0 and buffer exchanged toPBS. In some instances, instead of initiating conjugation with antibodyon the resin, the column was washed with at least 20 column volumes ofPBS, and antibody was eluted with IgG elution buffer and neutralizedwith buffer pH 8.0. Antibodies were then either used for conjugationreactions or flash frozen for future use.

In some instances, it is desired to conjugate to native Cys residues,such as those that usually form the heavy chain to light chaininterchain disulfide bond and the Cys residues in the hinge region ofthe antibody that usually form heavy chain to heavy chain interchaindisulfide bonds, in the absence of engineered Cys residues or at thesame time as conjugation was also directed to engineered Cys residues.In these cases, the antibody or antibody fragment was reduced by adding5-fold excess of TCEP to disulfide bonds and incubated the sample at 37°C. for 1 hour. The samples were then immediately conjugated or frozen at<−60° C. for future conjugation. Maleimide-containing compounds wereadded to antibodies or antibody fragments in PBS buffer (pH 7.2) atratios of typically 2:1 to Cys residues used for conjugation, andincubations were carried out for 1 hour. Typically, excess free compoundwas removed by desalting column followed by more extensive bufferexchange to PBS.

Conjugation to lysine residues can be prepared by reacting antibodies orantibody fragments with a linker-drug compound which comprises an aminereactive group, such as an NHS ester or a tetrafluorophenyl ester,(e.g., compound (7), SMCC-DM1, Sulfo-SPDB-DM4 or SPDB-DM4). By way ofexample, Compound (7) was conjugated to lysine residues on anti-HER2Fab-HC-E152C. Specifically, anti-HER2 Fab-HC-E152C was expressed bytransient transfection in HEK293 cells. Fab was captured from the mediaby capto-L (GE Healthcare) affinity purification, eluted in IgG ElutionBuffer (Pierce) and buffer exchanged to PBS by ultraconcentrator(Amicon). To the Fab solution (5.8 mg/ml) was added a 2-fold molarexcess of Compound (7). The mixture was incubated at room temperaturefor 30 minutes and then the mixture was quenched with 50 mM Tris pH 8.The resulting conjugate was then purified by preparative SEC in PBS.

Generation of Antibody Fragments from Full-Length Antibodies

In some instances, antibody fragments were generated by geneticmanipulation of the antibody heavy chain coding sequence, as describedabove, such that the product of expression was a fragment of anantibody. In other instances, antibodies were generated by enzymaticdigest of full-length antibodies.

To generate Fab fragments comprising residues 1-222 (EU numbering) of astarting antibody, the full antibody was treated with immobilized papainresin (ThermoFisher Scientific) according to manufacturer's protocol.Briefly, the immobilized papain resin is prepared by equilibrating in adigestion buffer of freshly dissolved 20 mM cysteine-HCl adjusted to pH7.0. The antibody is adjusted to approximately 10 mg/ml and bufferexchanged into the digestion buffer and added to resin at a ratio of 4mg IgG per ml resin and incubated at 37° C. for 5-7 hours. The resin isthen removed, and the antibody fragment is purified by either anappropriate affinity resin, for example the intact IgG and Fc fragmentare separated from the Fab fragment by binding to Protein A resin, orthe separation is conducted by size exclusion chromatography.

To generate F(ab′)₂ fragments comprising residues 1-236 (EU numbering)of the starting antibody, the full antibody was treated with aproteolytic enzyme. Briefly, the antibody is prepared in PBS atapproximately 10 mg/ml. The enzyme is added at a 1:100 weight/weightratio and incubated for 2 hours at 37° C. The antibody fragment ispurified by either an appropriate affinity resin, for example the intactIgG and Fc fragment are separated from the Fab′ fragment by binding toProtein A resin, or the separation is conducted by size exclusionchromatography.

Properties of Anti-cKit-Toxin Antibody and Antibody Fragment Conjugates

Antibody and antibody fragment conjugates were analyzed to determineextent of conjugation. A compound-to-antibody ratio was extrapolatedfrom LC-MS data for reduced and deglycosylated (where appropriate)samples. LC/MS allows quantitation of the average number of molecules oflinker-payload (compound) attached to an antibody in a conjugate sample.High pressure liquid chromatography (HPLC) separates antibody into lightand heavy chains, and under reducing conditions, separates heavy chain(HC) and light chain (LC) according to the number of linker-payloadgroups per chain. Mass spectral data enables identification of thecomponent species in the mixture, e.g., LC, LC+1, LC+2, HC, HC+1, HC+2,etc. From the average loading on the LC and HC chains, the averagecompound to antibody ratio can be calculated for an antibody conjugate.A compound-to-antibody ratio for a given conjugate sample represents theaverage number of compound (linker-payload) molecules attached to atetrameric antibody containing two light chains and two heavy chains.

Conjugates were profiled using analytical size-exclusion chromatography(AnSEC) on Superdex 200 10/300 GL (GE Healthcare) and/or Protein KW-8035 μm 300×8 mm (Shodex) columns; aggregation was analyzed based onanalytical size exclusion chromatography.

Preparation of Exemplary Anti-cKIT Fab-Toxin Conjugates

To generate anti-cKIT Fab′-toxin DAR4 conjugates or anti-Her2 Fab-toxinDAR4 control conjugate, 50 mg full IgG (WT, without introducedcysteines) was digested with a proteolytic enzyme. The F(ab′)₂ fragmentwas purified by SEC on a Superdex-S200 (GE Healthcare) column.Alternatively, to generate anti-HER2 control conjugates or anti-cKitFab′-toxin DAR4 conjugates, a vector encoding the Fab′ HC wasco-transfected with a vector encoding the Fab′ LC in CHO. The expressedFab′ was purified by capture on Protein G resin. The F(ab′)₂ or Fab′ wasreduced by addition of TCEP (5× excess to interchain disulfides) andimmediately reacted with a compound of the invention (2.5× excess tofree Cys residues). Reaction was monitored by RP-HPLC, and additional 1×equivalents of compound were added until reaction was completed. Freecompound was removed by PD10 desalting column (GE Healthcare). DAR wereexperimentally determined to be >3.9. Specific conjugates studiedfurther in the provided examples are listed in Table 2.

To generate anti-cKIT Fab-toxin DAR2 conjugates, a vector encoding theFab HC with an introduced Cys residue (HC 1-221 with E152C by EUnumbering) was co-transfected with a vector encoding the Fab LC with anintroduced Cys residue (kappa LC K107C, kappa LC S114C, or kappa LCE165C by EU numbering) in HEK293. To generate anti-Her2 Fab-toxin DAR2control conjugates, a vector encoding the Fab HC with an introduced Cysresidue (HC 1-222 with E152C by EU numbering, and a C-terminal His₆ tag(SEQ ID NO: 162)) was co-transfected with a vector encoding the Fab LCwith an introduced Cys residue (kappa LC K107C, kappa LC S114C, or kappaLC E165C by EU numbering) in HEK293. The expressed Fabs were purified bycapture on Capto-L resin (GE Healthcare) and elution with standard IgGElution Buffer (Thermo). Fabs were buffer exchanged to PBS using Amiconultra devices. Fabs were reduced with DTT and allowed to reoxidize atroom temperature. After reformation of the interchain disulfide bond,the Fabs were conjugated to Compound 6 (3× excess to free Cys residues).Reaction was allowed to proceed for 30 min at room temperature andmonitored by RP-HPLC with detection at 310 nm. Conjugated Fabs werepurified over protein A (anti-her2) or capto-L (anti-cKit) resins andwere washed with PBS+1% Triton X-100 and washed with extensive PBSbefore elution in IgG Elution Buffer. Fabs were then buffer exchanged toPBS using Amicon Ultra devices. Specific conjugates studied further inthe provided examples are listed in Table 2 below with experimentallydetermined DAR values.

To generate anti-cKIT F(ab′)₂-toxin DAR2 conjugates, a vector encodingthe HC with introduced Cys residues (E152C and S375C by EU numbering)was co-transfected with a vector encoding the Fab LC in CHO. To generateanti-Her2 F(ab′)₂-toxin DAR2 control conjugates, a vector encoding theHC with introduced Cys residues (E152C and S375C by EU numbering) wasco-transfected with a vector encoding the Fab LC in HEK293. Theexpressed IgGs were purified by capture on protein A or mabselectsureresin (GE Healthcare) and elution with standard IgG Elution Buffer(Thermo). Full IgGs were reduced with DTT at room temperature andreoxidized following removal of DTT as monitored by RP-HPLC. Thereoxidized IgGs were then digested with a proteolytic enzyme to generateF(ab′)₂ fragments. For anti-cKIT fragments, F(ab′)₂'s were bufferexchanged to PBS using Amicon ultra devices. For anti-HER2 fragment,F(ab′)₂ fraction was enriched by preparative HIC and then bufferexchanged to PBS using Amicon ultra devices. The F(ab′)₂'s wereconjugated to Compound 4 or Compound 5 (4× excess to free Cys residues).Reaction was allowed to proceed for 30 min at room temperature andmonitored by RP-HPLC with detection at 310 nm. Conjugated F(ab′)₂'s werepurified over capto-L (anti-cKit Ab3) resins and were washed with PBS+1%Triton X-100 and washed with extensive PBS before elution in IgG ElutionBuffer or by preparative SEC (anti-her2 and anti-cKIT Ab4). F(ab′)₂'swere then concentrated and buffer exchanged to PBS using Amicon Ultradevices. Specific conjugates studied further in the provided examplesare listed in Table 2 below with experimentally determined DAR values.

To generate anti-cKIT Fab-toxin DAR1 conjugates, a vector encoding theHC with introduced Cys residues (E152C by EU numbering) wasco-transfected with a vector encoding the Fab LC in HEK293. Theexpressed IgG was purified by capture on protein A resin (GE Healthcare)and elution with standard IgG Elution Buffer (Thermo). Full IgGs werereduced with DTT at room temperature and reoxidized following removal ofDTT as monitored by RP-HPLC. IgG was digested with immobilized papain(Thermo) to generate Fab fragment. Fabs were buffer exchanged to PBSusing Amicon ultra devices. Fabs were conjugated to Compound 4 (4×excess to free Cys residues). Reaction was allowed to proceed for 30 minat room temperature and monitored by RP-HPLC with detection at 310 nm.Conjugated Fab was purified by preparative SEC in PBS.

To generate anti-Her2 Fab-toxin DAR1 control conjugates, a vectorencoding the Fab HC with introduced Cys residues (E152C by EU numbering)was co-transfected with a vector encoding the Fab LC in HEK293. Theexpressed Fabs were purified by capture on Capto-L resin (GE Healthcare)and elution with standard IgG Elution Buffer (Thermo). Fabs were bufferexchanged to PBS using Amicon ultra devices. The Fab was conjugated toCompound 7 (2× molar excess to Fab). Reaction was allowed to proceed for30 min at room temperature and monitored by RP-HPLC with detection at310 nm. Conjugation was quenched with 50 mM Tris pH 8.0. Conjugated Fabwas purified by preparative SEC in PBS.

TABLE 2  Exemplary anti-cKIT or control conjugates Conjug. AntibodyConjugation Antibody fragment Antibody fragment No. fragment MethodHC sequence LC sequence Payload DAR J1 Anti-cKIT Native SEQ ID NO: 14SEQ ID NO: 25 Compound 4 Fab′1 Cysteine (1) conjugation* J2 Anti-cKITNative SEQ ID NO: 40 SEQ ID NO: 49 Compound 4 Fab′2 Cysteine (1)conjugation J3 Anti-cKIT Native SEQ ID NO: 58 SEQ ID NO: 25 Compound 4Fab′3 Cysteine (1) conjugation J4 Anti-cKIT Native SEQ ID NO: 73SEQ ID NO: 84 Compound 4 Fab′4 Cysteine (1) conjugation J5 Anti-cKITNative SEQ ID NO: 99 SEQ ID NO: 110 Compound 4 Fab′5 Cysteine (1)conjugation J6 Anti-Her2 Native EVQLVESGGGLVQ DIQMTQSPSSLSAS Compound 4Fab′ Cysteine PGGSLRLSCAASG VGDRVTITCRASQ (1) conjugation FNIKDTYIHWVRQADVNTAVAWYQQKP PGKGLEWVARIYP GKAPKLLIYSASFL TNGYTRYADSVKG YSGVPSRFSGSRSRFTISADTSKNTAY GTDFTLTISSLQPE LQMNSLRAEDTAV DFATYYCQQHYTT YYCSRWGGDGFYPPTFGQGTKVEIKR AMDYWGQGTLVT TVAAPSVFIFPPSD VSSASTKGPSVFPL EQLKSGTASVVCLLAPSSKSTSGGTAA NNFYPREAKVQWK LGCLVKDYFPEPVT VDNALQSGNSQES VSWNSGALTSGVHVTEQDSKDSTYSL TFPAVLQSSGLYSL SSTLTLSKADYEKH SSVVTVPSSSLGT KVYACEVTHQGLSQTYICNVNHKPSNT SPVTKSFNRGEC KVDKKVEPKSCDK (SEQ ID NO: 147) THTCPPCPAPELLG (SEQ ID NO: 146) J7 Anti-cKIT Engineered SEQ ID NO: 130 SEQ ID NO: 134Compound 1.7 Fab3 Cysteines at (6) HC-E152C and LC- E1650 (EU) J8Anti-Her2 Engineered EVQLVESGGGLVQ DIQMTQSPSSLSAS Compound 1.8 FabCysteines at PGGSLRLSCAASG VGDRVTITCRASQ (6) HC-E152C FNIKDTYIHWVRQADVNTAVAWYQQKP and LC- PGKGLEWVARIYP GKAPKLLIYSASFL K1070 (EU)TNGYTRYADSVKG YSGVPSRFSGSRS RFTISADTSKNTAY GTDFTLTISSLQPE LQMNSLRAEDTAVDFATYYCQQHYTT YYCSRWGGDGFY PPTFGQGTKVEICR AMDYWGQGTLVT TVAAPSVFIFPPSDVSSASTKGPSVFPL EQLKSGTASVVCLL APSSKSTSGGTAA NNFYPREAKVQWK LGCLVKDYFPCPVVDNALQSGNSQES TVSWNSGALTSGV VTEQDSKDSTYSL HTFPAVLQSSGLY SSTLTLSKADYEKHSLSSVVTVPSSSLG KVYACEVTHQGLS TQTYICNVNHKPSN SPVTKSFNRGEC TKVDKKVEPKSCD(SEQ ID NO: 149) KHHHHHH (SEQ ID NO: 148) J9 Anti-cKIT EngineeredQVQLQQSGPGLVK SEQ ID NO: 84 Compound 1.9 F(ab′4)₂ Cysteines atPSQTLSLTCAISGD (4) HC-E1520 SVSTNSAAWNWIR (EU) QSPSRGLEWLGRIYYRSQWLNDYAVS VKSRITINPDTSKN QFSLQLNSVTPED TAVYYCARQLTYP YTVYHKALDVWGQGTLVTVSSastkgpsv fplapsskstsggtaalgcl vkdyfpCpvtvswnsgaltsgvhtfpavlqssglysls svvtvpssslgtqtyicnvn hkpsntkvdkrvepkscdkthtcppcpapellg (SEQ ID NO: 153) J10 Anti-cKIT Engineered QVQLVQSGAEVKKSEQ ID NO: 25 Compound 2 F(ab′3)₂ Cysteines at PGSSVKVSCKASG (5)HC-E1520 GTFSSYAISWVRQ (EU) APGQGLEWMGTIG PFEGQPRYAQKFQ GRVTITADESTSTAYMELSSLRSEDTA VYYCARGGYISDF DVWGQGTLVTVSS astkgpsvfplapsskstsggtaalgclykdyfpCpvt vswnsgaltsgyhtfpayl qssg lysIssyytypsssIgtqtyicnvn hkpsntkvdk rvepkscdkthtcppcpa pellg (SEQ ID NO: 154) J11Anti-cKIT Engineered QVQLVQSGAEVKK SEQ ID NO: 25 Compound 0.9 Fab3Cysteines at PGSSVKVSCKASG (4) HC-E152C GTFSSYAISWVRQ (EU) APGQGLEWMGTIGPFEGQPRYAQKFQ GRVTITADESTSTA YMELSSLRSEDTA VYYCARGGYISDF DVWGQGTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGC LVKDYFPcPVTVS WNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKV DKKVEPKSCDKTH (SEQ ID NO: 155) J12Anti-Her2 Engineered EVQLVESGGGLVQ SEQ ID NO: 147 Compound 1.7 F(ab′)2Cysteines at PGGSLRLSCAASG (4) HC-E152C FNIKDTYIHWVRQA (EU)PGKGLEWVARIYP TNGYTRYADSVKG RFTISADTSKNTAY LQMNSLRAEDTAV YYCSRWGGDGFYAMDYWGQGTLVT VSSASTKGPSVFPL APSSKSTSGGTAA LGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLG TQTYICNVNHKPSN TKVDKKVEPKSCD KTHTCPPCPAPELLG (SEQ ID NO: 156) J13 Anti-Her2 Engineered EVQLVESGGGLVQ SEQ ID NO: 147Compound 0.9 Fab Cysteines at PGGSLRLSCAASG (7) FNIKDTYIHWVRQA HC-E1520PGKGLEWVARIYP (EU) TNGYTRYADSVKG RFTISADTSKNTAY LQMNSLRAEDTAVYYCSRWGGDGFY AMDYWGQGTLVT VSSASTKGPSVFPL APSSKSTSGGTAA LGCLVKDYFPCPVTVSWNSGALTSGV HTFPAVLQSSGLY SLSSVVTVPSSSLG TQTYICNVNHKPSN TKVDKKVEPKSCDK (SEQ ID NO: 157) J14 Anti-cKIT Native SEQ ID NO: 14 SEQ ID NO: 25mc-MMAF 4 Fab′1 Cysteine conjugation J15 Anti-cKIT Native SEQ ID NO: 14SEQ ID NO: 25 Compound 4 Fab′1 Cysteine (5) conjugation J16 Anti-cKITNative SEQ ID NO: 14 SEQ ID NO: 25 Compound 4 Fab′1 Cysteine (2)conjugation J17 Anti-cKIT Native SEQ ID NO: 40 SEQ ID NO: 49 mc-MMAF 4Fab′2 Cysteine conjugation J18 Anti-cKIT Native SEQ ID NO: 40SEQ ID NO: 49 Compound 4 Fab′2 Cysteine (5) conjugation J19 Anti-cKITNative SEQ ID NO: 40 SEQ ID NO: 49 Compound 4 Fab′2 Cysteine (2)conjugation J20 Anti-cKIT Native SEQ ID NO: 132 SEQ ID NO: 25 mc-MMAF 4Fab′3 Cysteine conjugation J21 Anti-cKIT Native SEQ ID NO: 58SEQ ID NO: 25 Compound 4 Fab′3 Cysteine (5) conjugation J22 Anti-cKITNative SEQ ID NO: 132 SEQ ID NO: 25 Compound 4 Fab′3 Cysteine (2)conjugation Anti-cKIT HC-E152C QVQLQQSGPGLVK SEQ ID NO: 84 none Fab4(EU) PSQTLSLTCAISGD SVSTNSAAWNWIR QSPSRGLEWLGRI YYRSQWLNDYAVSVKSRITINPDTSKN QFSLQLNSVTPED TAVYYCARQLTYP YTVYHKALDVWGQGTLVTVSSastkgpsv fplapsskstsggtaalgcl ykdyfpCpytyswnsgaltsgvhtfpavlqssg lysis syytypsssIgtqtyicnvn hkpsntkvdkrvepkscdk (SEQ ID NO: 158) Anti-cKIT HG-E152C QVQLVQSGAEVK SEQ ID NO: 25 noneFab1 (EU) KPGSSVKVSCKA SGGTFSSYAISW VRQAPGQGLEW MGVIFPAEGAPGYAQKFQGRVTIT ADESTSTAYMEL SSLRSEDTAVYY CARGGYISDFDV WGQGTLVTVSSastkgpsvfplapssksts ggtaalgclvkdyfpCp vtvswnsgaltsgvhtfpavlqssglysIssvvtvp ssslgtqtyicnvnhkps ntkvdkrvepkscdk (SEQ ID NO: 159)Anti-cKIT HG-E152G QVQLVQSGAEVK SEQ ID NO: 49 none Fab2 (EU)KPGSSVKVSCKA SGGTFSSHALSW VRQAPGQGLEW MGGIIPSFGTADY AQKFQGRVTITADESTSTAYMELS SLRSEDTAVYYC ARGLYDFDYWG QGTLVTVSSastkg psvfplapsskstsggtaalgclvkdyfpCpvtv swnsgaltsgvhtfpavl qssglyslssvvtvpsssIgtqtyicnvnhkpsntk vdkrvepkscdk (SEQ ID NO: 160) Anti-cKIT HG-E152GQVQLVQSGAEVK SEQ ID NO: 25 none Fab3 (EU) KPGSSVKVSCKA SGGTFSSYAISWVRQAPGQGLEW MGTIGPFEGQPR YAQKFQGRVTIT ADESTSTAYMEL SSLRSEDTAVYYCARGGYISDFDV WGQGTLVTVSSA STKGPSVFPLAP SSKSTSGGTAAL GCLVKDYFPCPVTVSWNSGALTSG VHTFPAVLQSSG LYSLSSVVTVPSS SLGTQTYICNVN HKPSNTKVDKKVEPKSCDK (SEQ ID NO: 161) Anti-Her2 HC-E152C SEQ ID NO: 157SEQ ID NO: 147 none Fab (EU) Anti-cKIT Native SEQ ID NO: 143SEQ ID NO: 110 Compound 3.9 Fab′5 Cysteine (1) conjugation Anti-cKITNative SEQ ID NO: 143 SEQ ID NO: 110 Compound 3.7 Fab′5 Cysteine (1)conjugation Anti-cKIT Native SEQ ID NO: 143 SEQ ID NO: 110 mc-MMAF 4Fab′5 Cysteine conjugation JW Anti-cKIT Native SEQ ID NO: 126SEQ ID NO: 49 Compound 3.9 Fab′2 Cysteine (1) conjugation JX Anti-cKITNative SEQ ID NO: 126 SEQ ID NO: 49 mc-MMAF 3.9 Fab′2 Cysteineconjugation JY Anti-cKIT Native SEQ ID NO: 132 SEQ ID NO: 25 Compound3.9 Fab′3 Cysteine (1) conjugation JZ Anti-cKIT Native SEQ ID NO: 132SEQ ID NO: 25 mc-MMAF 3.9 Fab′3 Cysteine conjugation * native Cysconjugation means the drug is attached to the antibody fragment at oneor more of the native cysteine residues selected from LC-214C andHC-220C-226C-229C (all positions by EU numbering).

Example 2: Generation of Human, Cyno, Mouse and Rat cKIT ExtracellularDomain Proteins, and of cKIT Subdomains 1-3, and 4-5 for Binding Assays

Human, mouse and rat cKIT extracellular domains (ECD) were genesynthesized based on amino acid sequences from the GenBank or Uniprotdatabases (see Table 3 below). Cynomolgus cKIT and 1 ECD cDNA templatewere gene synthesized based on amino acid sequences informationgenerated using mRNA from various cyno tissues (e.g. ZyagenLaboratories; Table 4 below). All synthesized DNA fragments were clonedinto appropriate expression vectors e.g. hEF1-HTLV based vector(pFUSE-mIgG2A-Fc2) with C-terminal tags to allow for purification.

TABLE 3  Sequences of human, mouse, rat cKIT constructs Accession SEQ IDName Description Number NO: Human cKITHuman cKIT tr. variant 2 , residues NM_001093772 112 D1-5 26-520-TAG(extracellular QPSVSPGEPSPPSIHPGKSDLIVRVGDEI domain)RLLCTDPGFVKWTFEILDETNENKQNEW ITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLT DPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSV LSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTVVKRENSQ TKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVV DKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKS ENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVN GMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSI DSSAFKHNGTVECKAYNDVGKTSAYFNFAFKEQIHPHTLFTPRSHHHHHH Human cKIT Human cKIT tr. Variant 1, residuesNM_000222 113 D1-3 26-311-TAG QPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEW ITEKAEATNTGKYTCTNKHGLSNSIYVFV R DPAKLFLVDRSLYGKEDNDTLVRCPLT DPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVD QEGKSV LSE KFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTVVKRENSQ TKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVV DKGRSHHHHHH Human cKITHuman cKIT tr. variant 1, residues NM_000222 114 D4-5 311-524-TAGGFINIFPMINTTVFVNDGENVDLIVEYEAF PKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSN SDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSAS VLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFK GNNKEQIHPHTLFTPRSHHHHHH Mouse cKITMouse cKIT tr. variant 1, residues NM_001122733 115 D1-5 26-527-TAGSQPSASPGEPSPPSIHPAQSELIVEAGDT LSLTCIDPDFVRWTFKTYFNEMVENKKNEWIQEKAEATRTGTYTCSNSNGLTSSIYV FVRDPAKLFLVGLPLFGKEDSDALVRCPLTDPQVSNYSLIECDGKSLPTDLTFVPNPK AGITIKNVKRAYHRLCVRCAAQRDGTWLHSDKFTLKVRAAIKAIPVVSVPETSHLLKK GDTFTVVCTIKDVSTSVNSMWLKMNPQPQHIAQVKHNSWHRGDFNYERQETLTISS ARVDDSGVFMCYANNTFGSANVTTTLKVVEKGFINISPVKNTTVFVTDGENVDLVVE YEAYPKPEHQQWIYMNRTSANKGKDYVKSDNKSNIRYVNQLRLTRLKGTEGGTYT FLVSNSDASASVTFNVYVNTKPEILTYDRLINGMLQCVAEGFPEPTIDWYFCTGAEQ RCTTPVSPVDVQVQNVSVSPFGKLVVQSSIDSSVFRHNGTVECKASNDVGKSSAFF NFAFKEQIQAHTLFTPLEVLFQGPRSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIK DVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSA LPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKK QVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKN WVERNSYSCSVVHEGLHNHHTTKSFSR TPGK Rat cKIT Rat cKIT, residues 25-526-TAG NM_022264 116 D1-5SQPSASPGEPSPPSIQPAQSELIVEAGDT IRLTCTDPAFVKWTFEILDVRIENKQSEWIREKAEATHTGKYTCVSGSGLRSSIYVFV RDPAVLFLVGLPLFGKEDNDALVRCPLTDPQVSNYSLIECDGKSLPTDLKFVPNPKA GITIKNVKRAYHRLCIRCAAQREGKWMRSDKFTLKVRAAIKAIPVVSVPETSHLLKEG DTFTVICTIKDVSTSVDSMWIKLNPQPQSKAQVKRNSWHQGDFNYERQETLTISSA RVNDSGVFMCYANNTFGSANVTTTLKVVEKGFINIFPVKNTTVFVTDGENVDLVVEF EAYPKPEHQQWIYMNRTPTNRGEDYVKSDNQSNIRYVNELRLTRLKGTEGGTYTFL VSNSDVSASVTFDVYVNTKPEILTYDRLMNGRLQCVAAGFPEPTIDWYFCTGAEQR CTVPVPPVDVQIQNASVSPFGKLVVQSSIDSSVFRHNGTVECKASNAVGKSSAFFNF AFKGNSKEQIQPHTLFTPRSLEVLFQGPGSPPLKECPPCAAPDLLGGPSVFIFPPKI KDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVV SALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMT KKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKST WERGSLFACSVVHEGLHNHLTTKTISRS LGK

TABLE 4  Sequences of cynomolgus cKIT proteinAmino acid sequence in one letter code, signal peptide underlined SEQCynomolgus monkey cKIT,  ID Construct residues 25-520-TAG NO CynomolgusMYRMQLLSCIALSLALVTNSQPSVSPGEPSPPS 117 monkeyIHPAKSELIVRVGNEIRLLCIDPGFVKWTFEIL cKIT D1-5DETNENKQNEWITEKAEATNTGKYTCTNKHGLS SSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTSYSLKGCQGKPLPKDLRFVPDPKAGI TIKSVKRAYHRLCLHCSADQEGKSVLSDKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIK DVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVT TTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENE SNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNASIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFP EPTIDWYFCPGTEQRCSASVLPVDVQTLNASGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKT SAYFNFAFKGNNKEQIHPHTLFTPRSHHHHHH

The desired cKIT recombinant proteins were expressed in HEK293 derivedcell lines (293FS) previously adapted to suspension culture and grown inserum-free medium FreeStyle-293 (Gibco, catalogue #12338018). Both smallscale and large scale protein production were via transient transfectionand was performed in multiple shaker flasks (Nalgene), up to 1 L each,with 293Fectin® (Life Technologies, catalogue #12347019) as a plasmidcarrier. Total DNA and 293Fectin was used at a ratio of 1:1.5 (w:v). DNAto culture ratio was 1 mg/L. The cell culture supernatants wereharvested 3-4 days post transfection, centrifuged and sterile filteredprior to purification.

Tapped ECD Protein Purification

Recombinant Fc-tagged cKIT extracellular domain proteins (e.g., humancKIT ECD-Fc, human cKIT (ECD subdomains 1-3, 4-5)-Fc, cyno cKIT-mFc, ratcKIT-mFc, mouse cKIT-mFc) were purified from the cell culturesupernatant. The clarified supernatant was passed over a Protein ASepharose® column which had been equilibrated with PBS. After washing tobaseline, the bound material was eluted with Pierce Immunopure® low pHElution Buffer, or 100 mM glycine (pH 2.7) and immediately neutralizedwith ⅛^(th) the elution volume of 1 M Tris pH 9.0. The pooled proteinwas concentrated if necessary using Amicon® Ultra 15 mL centrifugalconcentrators with 10 kD or 30 kD nominal molecular weight cut-offs. Thepools were then purified by SEC using a Superdex® 200 26/60 column toremove aggregates. The purified protein was then characterized bySDS-PAGE and SEC-MALLS (Multi-angle laser light scattering).Concentration was determined by absorbance at 280 nm, using thetheoretical absorption coefficients calculated from the sequence byVector NTI.

Example 3: Binding of cKIT Fabs to cKIT ECD Subdomains

To help define the binding sites of the cKIT Abs, the human cKIT ECD wasdivided into subdomains 1-3 (ligand binding domain) and subdomains 4-5(dimerization domain). To determine which subdomains were bound, asandwich ELISA assay was employed. 1 μg/ml of ECD diluted in 1×Phosphate buffered saline corresponding to cKIT subdomains 1-3,subdomains 4-5 or full-length cKIT ECD were coated on 96 well Immulon®4-HBX plates (Thermo Scientific Cat#3855, Rockford, Ill.) and incubatedovernight at 4° C. Plates were washed three times with wash buffer (1×Phosphate buffered saline (PBS) with 0.01% Tween-20 (Bio-Rad 101-0781)).Plates were blocked with 280 μl/well 3% Bovine Serum Albumin diluted in1×PBS for 2 hrs at room temperature. Plates were washed three times withwash buffer. Antibodies were prepared at 2 μg/ml in wash buffer with5-fold dilutions for 8 points and added to ELISA plates at 100 μl/wellin triplicate. Plates were incubated on an orbital shaker shaking at 200rpm for 1 hr at room temperature. Assay plates were washed three timeswith wash buffer. Secondary antibody F(ab′)₂ Fragment Goat anti-humanIgG (H+L) (Jackson Immunoresearch Cat#109-036-088, West Grove, Pa.) wasprepared 1:10,000 in wash buffer and added to ELISA plates at 100μl/well. Plates were incubated with secondary antibody for 1 hr at roomtemperature shaking at 200 rpm on an orbital shaker. Assay plates werewashed three times with wash buffer. To develop the ELISA signal, 100μl/well of Sure Blue® TMB substrate (KPL Cat#52-00-03, Gaithersburg,Md.) was added to plates and allowed to incubate for 10 mins at roomtemperature. To stop the reaction 50 μl of 1N Hydrochloric Acid wasadded to each well. Absorbance was measured at 450 nm using a MolecularDevices SpectraMax® M5 plate reader. To determine the binding responseof each antibody the optical density measurements were averaged,standard deviation values generated and graphed using Excel. The bindingcharacteristics of individual anti-cKIT antibody to cKIT can be found inTable 5.

Example 4: Affinity Measurements of cKIT Antibodies

Affinity of the antibodies to cKIT species orthologues and also to humancKIT was determined using SPR technology using a Biacore® 2000instrument (GE Healthcare, Pittsburgh, Pa.) and with CM5 sensor chips.

Briefly, HBS-P (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005% SurfactantP20) supplemented with 2% Odyssey® blocking buffer (Li-Cor Biosciences,Lincoln, Nebr.) was used as the running buffer for all the experiments.The immobilization level and analyte interactions were measured byresponse unit (RU). Pilot experiments were performed to test and confirmthe feasibility of the immobilization of the anti-human Fc antibody(Catalog number BR100839, GE Healthcare, Pittsburgh, Pa.) and thecapture of the test antibodies.

For kinetic measurements, the experiments were performed in which theantibodies were captured to the sensor chip surface via the immobilizedanti-human Fc antibody and the ability of the cKIT proteins to bind infree solution was determined. Briefly, 25 μg/ml of anti-human Fcantibody at pH 5 was immobilized on a CM5 sensor chip through aminecoupling at flow rate of 5 μl/min on both flow cells to reach 10,500RUs. 0.1-1 μg/ml of test antibodies were then injected at 10 μl/min for1 minute. Captured levels of the antibodies were generally kept below200 RUs. Subsequently, 3.125-50 nM of cKIT receptor extracellulardomains (ECD) were diluted in a 2-fold series and injected at a flowrate of 40 μl/min for 3 min over both reference and test flow cells. Atable of tested ECDs is listed below (Table 5). Dissociation of ECDbinding was followed for 10 min. After each injection cycle, the chipsurface was regenerated with 3 M MgCl₂ at 10 μl/min for 30 seconds. Allexperiments were performed at 25° C. and the response data were globallyfitted with a simple 1:1 interaction model (using Scrubber 2 ® softwareversion 2.0b (BioLogic Software) to obtain estimates of on rate (k_(a)),off-rate (k_(d)) and affinity (K_(D)). Table 6 lists the domain bindingand affinity of selected anti-cKIT antibodies.

TABLE 5 cKIT ECD isotype and source ECD Isotype Tag Source HumanC-terminal 6x His Novartis construct (SEQ ID NO: 162) Cyno C-terminal 6xHis Novartis construct (SEQ ID NO: 162) Mouse C-terminal 6x His SinoBiological Inc (SEQ ID NO: 162) (Catalog number: 50530-M08H) RatC-terminal mFc Novartis construct

TABLE 6 Antibody affinity and cross reactivity KD (pM) KD (pM) cKIT tohuman to cyno Reactivity Reactivity domain cKIT ECD cKIT ECD to mouse torat Ab binding in SET in SET cKIT cKIT Anti-cKIT D1-3 94 170 Notreactive Not reactive Ab1 Anti-cKIT D1-3 7 10 Not reactive Not reactiveAb2 Anti-cKIT D1-3 160 52 Not reactive Not reactive Ab3 Anti-cKIT D4-52400 140 Yes Yes Ab4 Anti-cKIT D1-3 110 180 Yes Yes Ab5

Example 5 In Vitro Human and Mouse HSC Cell Killing Assays by cKITADCsIn Vitro HSC Viability Assays

Human mobilized peripheral blood hematopoietic stem cells (HSCs) wereobtained from HemaCare (catalog number M001F-GCSF-3). Each vial of 1million cells was thawed and diluted into 10 ml of 1×HBSS andcentrifuged for 7 minutes at 1200 rpm. The cell pellet was resuspendedin 18 ml of growth medium containing three growth factors (StemSpan SFEM(StemCell Technologies, catalog number 09650) with 50 ng/ml each of TPO(R&D Systems, catalog number 288-TP) Flt3 ligand (Life Technologies,catalog number PHC9413), and IL-6 (Life Technologies, catalog numberPHC0063), supplemented with amino acids (Gibco, catalog number10378-016)).

Bone marrow cells from C57BL/6J mice were harvested from the femurs andtibiae, resuspended in IMDM (HyClone, catalog number SH30228.01) andpooled. Cells were centrifuged for 10 minutes at 300 g. The cell pelletwas resuspended in AutoMACS buffer (1×PBS+0.5% BSA+2 mM EDTA) at aconcentration of 100 million cells in 40 μl. The lineage antibodycocktail (Miltenyi, catalog number 130-090-858) was added at aconcentration of 10 ul per 100 million cells. Cells were incubated for10 minutes in the cold room before addition of 30 μl of AutoMACS bufferand 20 μl of biotinylated magnetic beads per 100 million cells. This newsuspension was incubated in the cold room for 15 minutes. Cells werecentrifuged for 10 minutes at 300 g. The pellet was resuspended in 2 mlof AutoMACS buffer and passed through a cell strainer. Cells wereselected on the AutoMACS using the “deplete” protocol. The negativefraction from the sort was centrifuged for 10 minutes at 300 g andresuspended in 1 ml of HBSS. The resuspended cells were stained withanti-CD45-PerCP-Cy5.5 (Becton Dickinson, catalog number 550994),anti-CD48-FITC (eBioscience, catalog number 11-0481-82), anti-CD150-PE(BioLegend, catalog number 115904), and anti-Sca-1 (Becton Dickinson,catalog number 560653). Cells were incubated at room temperature for 30minutes, centrifuged for 5 minutes at 300 g, and resuspended in 700 μlof FACS buffer for sorting. Sca-1+ cells were positively sorted on aFACS Aria. After sort, cells were placed into growth media containingthree growth factors (StemSpan SFEM with 50 ng/ml TPO (R&D Systems,catalog number 288-TP), Flt3 ligand (Life Technologies, catalog numberPHC9413), and IL-6 (Life Technologies, catalog number PHC0063)supplemented with amino acids (Gibco, catalog number 10378-016)).

Test agents were diluted in duplicate into a 384-well black assay plateat a final volume of 5 μl, starting at 10 μg/ml and with 1:3 serialdilutions. Cells from above were added to each well at a final volume of45 μl. Cells were incubated at 37° C. and 5% oxygen for 7 days. At theend of culture, cells were harvested for staining by centrifuging theassay plate for 4 minutes at 1200 rpm. Supernatants were then aspiratedand the cells were washed and transferred to a different 384-well plate(Greiner Bio-One TC-treated, black clear flat, catalog number 781092).

For human cell assays, each well was stained with anti-CD34-PerCP(Becton Dickinson, catalog number 340666) and anti-CD90-APC (BectonDickinson, catalog number 559869), washed, and resuspended in FACSbuffer to a final volume of 50 μl. For mouse cell assays, each well wasstained with anti-CD45-PerCP-Cy5.5 (Becton Dickinson, catalog number550994), anti-CD48-FITC (eBioscience, catalog number 11-0481-82),anti-CD150-PE (BioLegend, catalog number 115904), anti-cKIT-APC (BectonDickinson, catalog number 553356), and anti-Sca-1 (Becton Dickinson,catalog number 560653), washed, and resuspended in FACS buffer to afinal volume of 50 μl. Cells were then analyzed on a Becton DickinsonFortessa flow cytometer and quantified for analysis.

Toxin conjugates of antibodies and antibody fragments recognizing cKITkilled HSCs as determined in this assay. Quantitation of cells by FACSshowed fewer viable cells in wells treated with anti-cKIT-toxinconjugates than in control wells treated with PBS or with isotypecontrol toxin conjugates of antibody or antibody fragment. Data areshown in FIG. 1, FIG. 2 and FIG. 9 and summarized in Table 7. The namingconvention used herein is J#, correspond to the specific Conjugate No.described in Table 2.

TABLE 7 Cell viability after treatment of anti-cKIT Fab-toxin conjugatesConjugate tested Cell population EC50 (ng/ml) J3 Human total nucleatedcells 45 Human CD34+ cells 8 Human CD90+ cells 12 J2 Human totalnucleated cells 58 Human CD34+ cells 11 Human CD90+ cells 16 J1 Humantotal nucleated cells 48 Human CD34+ cells 11 Human CD90+ cells 13 J4Mouse total nucleated cells 3800 Mouse CD45+ cells 3800 Mouse cKIT+cells 8 J5 Mouse total nucleated cells 210 Mouse CD45+ cells 210 MousecKIT+ cells 120 J14 Human total nucleated cells 7 Human CD34+ cells 10Human CD90+ cells 11 J15 Human total nucleated cells 6 Human CD34+ cells5 Human CD90+ cells 1 J16 Human total nucleated cells 6 Human CD34+cells 7 Human CD90+ cells 9 J17 Human total nucleated cells 12 HumanCD34+ cells 16 Human CD90+ cells 37 J18 Human total nucleated cells 15Human CD34+ cells 11 Human CD90+ cells 3 J19 Human total nucleated cells14 Human CD34+ cells 16 Human CD90+ cells 23 J20 Human total nucleatedcells 20 Human CD34+ cells 25 Human CD90+ cells 72 J21 Human totalnucleated cells 154 Human CD34+ cells 88 Human CD90+ cells 22 J22 Humantotal nucleated cells 8 Human CD34+ cells 10 Human CD90+ cells 17

Example 6 In Vitro Assay of Human Mast Cell Degranulation

Mature mast cells were generated using CD34+ progenitors from mobilizedperipheral blood. CD34+ cells were cultured in StemSpan SFEM (StemCellTechnologies) supplemented with recombinant human stem cell factor(rhSCF, 50 ng/ml, Gibco), recombinant human interleukin 6 (rhIL-6, 50ng/ml, Gibco), recombinant human IL-3 (30 ng/ml, Peprotech), GlutaMAX (2nM, Gibco), penicillin (100 U/ml, Hyclone) and streptomycin (100 μg/ml,Hyclone). Recombinant hII-3 was added only during the first week of theculture. After the third week, half of the medium was replaced weeklywith fresh medium containing rhIL-6 (50 ng/ml) and rhSCF (50 ng/ml).Mature mast cell purity was evaluated by surface staining ofhigh-affinity IgE receptor (FCERI, eBioscience) and CD117 (BD). Cellswere used between week 8 and 12 of the culture.

The derived mast cells were washed once to remove SCF, and the requiredamount of cells was incubated overnight in mast cell medium containingrhIL-6 (50 ng/ml) with or without rhSCF (50 ng/ml). As positive controlfor mast cell degranulation, a portion of the cells were sensitized withhuman myeloma IgE (100 ng/ml, EMD Millipore). The following day,anti-cKIT antibody or antibody fragments or toxin conjugates thereof,mouse monoclonal anti-human IgG1 (Fab specific, Sigma), goat anti-humanIgE (Abcam) and compound 48/80 (Sigma) dilutions were prepared in HEPESdegranulation buffer (10 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.4 mMsodium phosphate dibasic, 5.6 mM glucose, pH adjusted at 7.4 and mixedwith 1.8 mM calcium chloride and 1.3 mM magnesium sulfate) supplementedwith 0.04% bovine serum albumin (BSA, Sigma). Test agents and anti-IgG1were mixed together in a V-bottom 384-well assay plate while anti-IgEand compound 48/80 were tested alone. The assay plate was incubated 30min at 37° C. During the incubation, cells were washed 3 times withHEPES degranulation buffer+0.04% BSA to remove medium and unbound IgE.Cells were resuspended in HEPES degranulation buffer+0.04% BSA andseeded at 3000 cells per well in the assay plate for a final reactionvolume of 50 μl. Cells that were sensitized with IgE were used only withanti-IgE as a positive control for degranulation. The assay plate wasincubated 30 min at 37° C. for degranulation to occur. During thisincubation, p-nitro-N-acetyl-β-D-glucosamine (pNAG, Sigma) buffer wasprepared by sonicating 3.5 mg/ml of pNAG in citrate buffer (40 mM citricacid, 20 mM sodium phosphate dibasic, pH 4.5). 3-hexosaminidase releasewas measured by mixing 20 μl of cell supernatant with 40 μl of pNAGsolution in a flat-bottom 384-well plate. This plate was incubated for1.5 hour at 37° C., and the reaction was stopped by the addition of 40μl of stop solution (400 mM glycine, pH 10.7). Absorbance was read usinga plate reader at A=405 nm with reference filter at A=620 nm.

Full-length IgG controls used in the mast cell degranulation assays aredescribed in Table 8.

TABLE 8 Full-length IgG controls used in the mast cell degranulation assays NameFIC sequence LC sequence IgG control Anti-cKIT SEQ ID NO: 12SEQ ID NO: 25 for J1 Ab1 IgG control Anti-cKIT SEQ ID NO: 38SEQ ID NO: 49 for J2 Ab2 IgG control Anti-cKIT SEQ ID NO: 56SEQ ID NO: 25 for J3 Ab3 IgG control Anti-cKIT SEQ ID NO: 71SEQ ID NO: 84 for J4 Ab4 IgG control Anti-Her2 EVQLVESGGGLVQPGGSLRLSCAASSEQ ID NO: 147 for J6 GFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 150)

As shown in FIG. 3A, certain clones of the antagonist anti-cKIT antibodyclass are less likely to cause mast cell degranulation. It furthersuggests that the Fab or Fab′ fragment is not able to cause mast celldegranulation. Upon crosslinking with a Fab-specific antibody (FIG.3C-3J), the full length IgG but not the anti-cKIT Fab′-toxin DAR4 formatshows increased degranulation. This suggests that the Fab′ fragment doesnot cause mast cell degranulation even when bound and multimerized intolarger complexes as could be observed if a patient developed or hadpre-existing anti-drug antibodies recognizing Fab or Fab′ fragments.

Example 7 In Vivo Ablation of Human HSCs from Mouse Host

To assess test agents for in vivo efficacy against human HSCs, mice thatare severely immune deficient (NOD.Cg-Prkdc^(scid) IL2rg^(tm1wJ)/SzJ,Jackson Laboratory, stock number 005557, a.k.a. NSG) were transplantedwith human HSCs after sublethal irradiation (250 RADS in a ¹³⁷Cs gammairradiator). CDs34+ hematopoietic stem cells (HSCs) were obtained fromAIICells (catalog numberCB008F-S). Each vial of 1 million cells wasthawed, diluted into 10 ml 1×HBSS and centrifuged for 7 minutes at 1200rpm. The cell pellet was resuspended in HBSS at 100,000 cells/ml. Atotal of 20,000 cells were transplanted per mouse via retro orbitalinjection 24 hours after irradiation. Human HSCs were allowed to engraftin the NSG mice for at least 4 weeks. Percent human chimerism wasdetermined by flow cytometry of blood samples. For this, blood wasstained with the following antibodies: anti-human CD45-e450(eBioscience, catalog #48-0459-42), anti-mouse CD45-APC (BectonDickinson, catalog #559864anti-human anti-human CD33-Pe (BectonDickinson, catalog #347787), anti-human CD19-FITC (Becton Dickinson,catalog #555422), and anti-human CD3-PeCy7 (Becton Dickinson, catalog#557851). Once human chimerism was confirmed, humanized NSG mice weredosed with a test agent intraperitoneally b.i.d. The degree of humanchimerism was re-assessed after dosing. To assess presence or absence ofhuman HSCs, mice were euthanized and bone marrow was isolated andstained with the following antibodies: anti-human CD45-e450(eBioscience, catalog #48-0459-42), anti-mouse CD45-APC (BectonDickinson, catalog #559864), anti-human CD34-PE (Becton Dickinson,catalog #348057), anti-human CD38-FITC (Becton Dickinson, catalog#340926), anti-human CD11b-PE (Becton Dickinson, catalog #555388),anti-human CD33-PeCy7 (Becton Dickinson, catalog #333946), anti-humanCD19-FITC (Becton Dickinson, catalog #555412), and anti-human CD3-PeCy7(Becton Dickinson, catalog #557851). Cell populations were assessed viaflow cytometry and analyzed with FlowJo.

In one particular experiment, mice were dosed with 10 mg/kg of conjugateJ7 (described in Table 2) twice a day for 1 2, or 4 days or isotypecontrol conjugate J8 twice a day for 4 days. Mice were euthanized on day21 and their bone marrow was analyzed. As shown in FIG. 4, mice treatedwith conjugate J7 for even 1 day showed depletion of human HSCs (humanCD45+, human CD34+, human CD38-), while mice treated with isotypecontrol conjugate J8 showed variable chimerism likely due to variationof the humanization prior to treatment, based on comparison to thevehicle (PBS) treated group.

In one particular experiment, mice were dosed with 10 mg/kg of anti-cKITconjugate J1, J2, or J3, or isotype control conjugate J6 for 2 days.Mice were euthanized on day 21 and their bone marrow was analyzed. Asshown in FIG. 5, mice treated with an anti-cKIT conjugate J1, J2, or J3showed reduced human HSCs (human CD45+, human CD34+, human CD38-), whilemice treated with isotype control conjugate J6 showed variablechimerism.

In one particular experiment, mice were dosed with 10 mg/kg of anti-cKITconjugate JW, JX, JY, or JZ for 2 days. Mice were euthanized on day 21and their bone marrow was analyzed. As shown in FIG. 13, mice treatedwith an anti-cKIT conjugate JW, JX, JY, or JZ showed reduced human HSCs(human CD45+, human CD34+, human CD38-).

These three experiments together show that anti-cKIT Fab-toxin oranti-cKit Fab′-toxin conjugates were able to deplete HSCs from bonemarrow. Both the anti-cKIT Fab-amanitin conjugates (e.g., J7) and theanti-cKIT Fab′-auristatin conjugates (e.g., J1, J2, J3, JW, JX, JY) wereable to ablate human HSCs in vivo.

Example 8 In Vivo Ablation of Mouse HSCs in Immune-Competent Mice

To assess test agents for in vivo efficacy against mouse HSCs, C57BL/6Jmice (males, 10 weeks of age, Jackson Laboratory, stock #000664) weredosed with a test agent intraperitoneally b.i.d. Hematology profile wasassessed as early as one day after last dose or up to 21 days after lastdose by standard methods. To assess presence or absence of mouse HSCs,mice were euthanized and bone marrow was isolated and stained with thefollowing antibodies: anti-mouse CD45-PerCP-Cy5.5 (Becton Dickinson,catalog #550994), anti-mouse cKIT-APC (Becton Dickinson, catalog#553356), anti-mouse CD48-FITC (eBioscience, catalog #11-0481-82),anti-mouse CD150-PE (BioLegend, catalog #115904), anti-mouse Sca-V450(Becton Dickinson, catalog #560653), anti-mouse Lin-biotin (Miltenyi,catalog #120-001-547), and Pe-Cy7-streptavidin (Becton Dickinson,catalog #557598). Cell populations were assessed via flow cytometry andanalyzed with FlowJo.

In one particular experiment, mice were dosed twice daily with 10 mg/kgof anti-cKIT conjugate J4 or J5 (described in Table 2), or PBS for 4days. Mice were euthanized on day 13 for bone marrow analysis. Groupstreated with anti-cKIT conjugate J4 or J5 showed significantly reducedlevels of stem and progenitor (cKIT+) cells in bone marrow compared toPBS treated control group (FIG. 6), demonstrating that treatment withanti-cKIT Fab′-auristatin conjugates such as J4 or J5 were able toablate HSCs in vivo in normal mice.

In order to determine if ablation by anti-cKIT antibody or antibodyfragment conjugates of the invention is sufficient to enable transplant,mice treated as above can be subsequently given HSC transplant. Forexample, CD45.2 mice treated with anti-cKIT Fab′-toxin conjugate can betransplanted with donor HSCs that are from CD45.1 mice approximately oneweek after dosing. In another example, mice treated with anti-cKITFab′-toxin conjugate can be treated with an immune-suppressing agent,such as an agent causing T-cell depletion approximately one week afterdosing and approximately 1-2 days prior to being transplanted with donorHSCs that are from CD45.1 mice. One method for T-cell depletion is todose mice with 0.5 mg per mouse of anti-mouse TCR P chain antibody(clone H57-597; Biolegend) q.i.d. for two days. T-cell depletion can beconfirmed by taking a blood sample for hematology profiling followingdosing. In such examples, the progress of the transplant would bemonitored by looking for CD45.1 chimerism in blood samples. In suchexamples, the success of transplant can be determined by euthanizing themice approximately 3-4 months or 5-6 months after transplant for bonemarrow analysis looking for populations of CD45.1 and CD45.2 HSCs.Alternatively, successful transplant can be determined by euthanizingthe mice at least 4 or 6 months after primary transplant and performingsecondary transplant into a fully irradiated host mice and looking forCD45.1 chimerism in blood samples following the secondary transplant.

Example 9 In Vitro Assay of Human Mast Cell Degranulation by Full-LengthAnti-cKIT Antibody, F(Ab′)₂ and Fab Fragments Thereof, and ConjugatesThereof

Mature mast cells were generated and tested with anti-cKIT antibody andF(ab′)₂ and Fab fragments or toxin conjugates thereof as described inExample 6.

As shown in FIGS. 7A-7C, full length anti-cKIT Ab4 (HC-E152C-S375C) andF(ab′4)₂ (HC-E152C) fragment conjugated with compound (4) caused mastcell degranulation when cross-linked, while no mast cell degranulationwas triggered by Fab4 (HC-E152C) fragment at all tested concentrations.FIGS. 7D-7F show that full length anti-cKIT Ab3 (HC-E152C-S375C) andF(ab′3)₂ (HC-E152C) fragment conjugated with compound (3) caused mastcell degranulation when cross-linked, while no mast cell degranulationwas triggered by Fab3 (E152C) fragment conjugated with compound (4) atall tested concentrations. This suggests that the Fab fragment orconjugates thereof do not cause mast cell degranulation even when boundand multimerized into larger complexes as could be observed if a patientdeveloped or had pre-existing anti-drug antibodies recognizing Fabfragments. On the other hand, F(ab′)₂ fragments and conjugates do causemast cell degranulation at a level similar to the full-length anti-cKITantibody when bound and multimerized into larger complexes.

Example 10 In Vitro Assay of Human Mast Cell Degranulation byFull-Length Anti-cKIT Antibody and F(Ab′₂ and Fab Fragments Thereof

Mature mast cells were generated and tested with anti-cKIT antibody andF(ab′)₂ and Fab fragments as described in Example 6.

As shown in FIGS. 8A-8C, full length anti-cKIT Ab4 and F(ab′4)₂ fragmentcaused mast cell degranulation when cross-linked, while no mast celldegranulation was triggered by Fab4 (HC-E152C) fragment at all testedconcentrations. FIGS. 8D-8F show that full length anti-cKIT Ab1 andF(ab′1)₂ fragment caused mast cell degranulation when cross-linked,while no mast cell degranulation was triggered by Fab1 (HC-E152C)fragment at all tested concentrations. FIGS. 8G-8I show that full lengthanti-cKIT Ab2 and F(ab′2)₂ fragment caused mast cell degranulation whencross-linked, while no mast cell degranulation was triggered by Fab2(HC-E152C) fragment at all tested concentrations. FIGS. 8J-8L show thatfull length anti-cKIT Ab3 and F(ab′3)₂ fragment caused mast celldegranulation when cross-linked, while no mast cell degranulation wastriggered by Fab3 (HC-E152C) fragment at all tested concentrations. Thissuggests that the Fab fragments do not cause mast cell degranulationeven when bound and multimerized into larger complexes as could beobserved if a patient developed or had pre-existing anti-drug antibodiesrecognizing Fab fragments. On the other hand, F(ab′)₂ fragments do causemast cell degranulation at a level similar to the full-length anti-cKITantibody when bound and multimerized into larger complexes.

Example 11 Syngeneic Bone Marrow Transplant in Immune-Competent Mice

In a particular experiment, C57BL/6J (CD45.2, males, 10 weeks of age,Jackson Laboratory, stock #000664) mice were dosed with 2.5, 5, or 10mg/ml anti-c-KIT Fab′5-(1) (Fab′-DAR4) in a mini-pump (Alzet, catalog#2001) placed subcutaneously in the back. The mini-pump held a volume of200 ul and infused at a constant rate of 1 ul/hour, over the course of 7days. Seven days after the mini-pump was implanted, it was then removedto halt any further administration of drug. Forty-eight hours aftermini-pump removal, the mice were transplanted with bone marrow fromdonor mice which were congenically marked at CD45 (CD45.1,B6.SJL-Ptprc^(a) Pepc^(b)/BoyJ, males, 10 weeks of age, JacksonLaboratory, stock #002014). The progress of the transplant was monitoredin the peripheral blood by looking for CD45.1 chimerism at one monthintervals. In this experiment, the blood chimerism has been monitoredfor 4 months as shown in FIG. 10. Chimerism in peripheral blood wasassessed by flow cytometry. Blood samples were stained withanti-mCD45.1-PerCP-Cy5.5 (1:100, BD #560580), anti-mCD45.2-BUV395(1:100, BD #564616), anti-Mac-PE (1:500, BD #553331), anti-GR1-FITC(1:100, BD #553127), anti-B220-APC (1:400, BD #553092), andanti-CD3-V450 (1:100, BD #560801) acquired on a Fortessa flow cytometer(Becton Dickinson), and analyzed with FlowJo software (TreeStar). Thelevel of chimerism was determined by comparing populations that werepositive for either CD45.2 (host) or CD45.1 (donor). Total donorchimerism represented the overall white blood cell population. Inaddition, subpopulations of T cells, B cells, and myeloid cells werefurther assessed for donor chimerism. T cell donor chimerism was basedon looking at CD45.2 (host) vs. CD45.1 (donor) in CD3+ cells. B celldonor chimerism was based on looking at CD45.2 (host) vs. CD45.1 (donor)in CD45R+ cells. Myeloid chimerism was based on looking at CD45.2 (host)vs. CD45.2 (donor) in Mac-1+/Gr-1+ cells. This experiment shows thatmice conditioned with an anti-cKit-Fab′ conjugate can be engrafted withdonor cells that reconstitute the myeloid, B-cell, and T-cell lineageswhich is indicative of successful HSC engraftment.

In another experiment, C57BL/6J (CD45.2, males, 10 weeks of age, JacksonLaboratory, stock #000664) mice were dosed with 2.5, 5, or 10 mg/mlanti-c-KIT Fab′5-(1) (Fab′-DAR4) or 10 mg/ml anti-c-KIT Fab′5-mc-MMAF(Fab′-DAR4) in a mini-pump (Alzet, catalog #2001) placed subcutaneouslyin the back. The mini-pump held a volume of 200 ul and infused at aconstant rate of 1 ul/hour, over the course of 7 days. Five days afterthe mini-pump was implanted, it was then removed to halt any furtheradministration of drug. Within 24 hours hours after mini-pump removal,the mice were then transplanted with bone marrow from donor mice whichwere congenically marked at CD45 (CD45.1, B6.SJL-Ptprc^(a)Pepc^(b)/BoyJ,males 10 weeks of age, Jackson Laboratory, stock #002014). The progressof the transplant was monitored in the peripheral blood by looking forCD45.1 chimerism at one month intervals. In this experiment, the bloodchimerism has been monitored for 2 months as shown in FIG. 11. Chimerismin peripheral blood was assessed by flow cytometry. Blood samples werestained with (anti-mCD45.1-PerCP-Cy5.5 (1:100, BD #560580),anti-mCD45.2-BUV395 (1:100, BD #564616), anti-Mac-PE (1:500, BD#553331), anti-GR1-FITC (1:100, BD #553127), anti-B220-APC (1:400, BD#553092), and anti-CD3-V450 (1:100, BD #560801)), acquired on a Fortessaflow cytometer (Becton Dickinson), and analyzed with FlowJo software(TreeStar). The level of chimerism was determined by comparingpopulations that were positive for either CD45.2 (host) or CD45.1(donor). Total donor chimerism represented the overall white blood cellpopulation. In addition, myeloid cells in the peripheral blood werefurther assessed for donor chimerism. Myeloid chimerism was based onlooking at CD45.2 (host) vs. CD45.2 (donor) in Mac-1+/Gr-1+ cells. Thisexperiment shows that mice conditioned with an anti-cKit-Fab′ conjugatecan be successfully engrafted with donor cells that reconstitute themyeloid compartment to an extent similar to mice conditioned with lethalirradiation (FIG. 11B). Lower levels of total donor chimerism inanti-cKit conditioned mice relative to irradiated mice (FIG. 11A) arelikely due to the more targeted nature of the anti-cKit conditioningagent that does not remove long-lived CD45.2+ B-cells and T-cells fromcirculation.

In another experiment, C57BL/6J (CD45.2, males, 10 weeks of age, JacksonLaboratory, stock #000664) mice were irradiated with 300 RADS. Afterthree days they were dosed with 2.5 or 5 mg/ml anti-c-KIT Fab′5-(1)(Fab′-DAR4) in a mini-pump (Alzet, catalog #2001) placed subcutaneouslyin the back. One group was treated only with irradiation and one groupwas treated only with 2.5 mg/ml anti-c-KIT Fab′5-(1) (Fab′-DAR4). Themini-pump held a volume of 200 ul and infused at a constant rate of 1ul/hour, over the course of 7 days. Three days after the mini-pump wasimplanted, it was then removed to halt any further administration ofdrug. Within 48 hours hours after mini-pump removal, the mice were thentransplanted with bone marrow from donor mice which were congenicallymarked at CD45 (CD45.1, B6.SJL-Ptprc^(a) Pepc^(b)/BoyJ, males 10 weeksof age, Jackson Laboratory, stock #002014). The progress of thetransplant was monitored in the peripheral blood by looking for CD45.1chimerism at one month intervals. In this experiment, the bloodchimerism has been monitored for 4 months as shown in FIG. 12. Chimerismin peripheral blood was assessed by flow cytometry. Blood samples werestained with (anti-mCD45.1-PerCP-Cy5.5 (1:100, BD #560580),anti-mCD45.2-BUV395 (1:100, BD #564616), anti-Mac-PE (1:500, BD#553331), anti-GR1-FITC (1:100, BD #553127), anti-B220-APC (1:400, BD#553092), and anti-CD3-V450 (1:100, BD #560801)), acquired on a Fortessaflow cytometer (Becton Dickinson), and analyzed with FlowJo software(TreeStar). The level of chimerism was determined by comparingpopulations that were positive for either CD45.2 (host) or CD45.1(donor). Total donor chimerism represented the overall white blood cellpopulation. In addition, myeloid cells in the peripheral blood werefurther assessed for donor chimerism. Myeloid chimerism was based onlooking at CD45.2 (host) vs. CD45.2 (donor) in Mac-1+/Gr-1+ cells. Thisexperiment shows that the anti-cKit-Fab′ conjugate can be used as aconditioning agent in combination with other agents such as low doseirradiation and that mice conditioned in this way can be successfullyengrafted with donor cells.

Unless defined otherwise, the technical and scientific terms used hereinhave the same meaning as that usually understood by a specialistfamiliar with the field to which the disclosure belongs.

Unless indicated otherwise, all methods, steps, techniques andmanipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks and the general background art mentioned herein andto the further references cited therein. Unless indicated otherwise,each of the references cited herein is incorporated in its entirety byreference.

Claims to the invention are non-limiting and are provided below.

Although particular aspects and claims have been disclosed herein indetail, this has been done by way of example for purposes ofillustration only, and is not intended to be limiting with respect tothe scope of the appended claims, or the scope of subject matter ofclaims of any corresponding future application. In particular, it iscontemplated by the inventors that various substitutions, alterations,and modifications may be made to the disclosure without departing fromthe spirit and scope of the disclosure as defined by the claims. Thechoice of nucleic acid starting material, clone of interest, or librarytype is believed to be a matter of routine for a person of ordinaryskill in the art with knowledge of the aspects described herein. Otheraspects, advantages, and modifications considered to be within the scopeof the following claims. Those skilled in the art will recognize or beable to ascertain, using no more than routine experimentation, manyequivalents of the specific aspects of the invention described herein.Such equivalents are intended to be encompassed by the following claims.Redrafting of claim scope in later filed corresponding applications maybe due to limitations by the patent laws of various countries and shouldnot be interpreted as giving up subject matter of the claims.

What is claimed is:
 1. A conjugate of Formula (I) or a pharmaceuticallyacceptable salt thereof;A-(LB-(D)_(n))_(y)   Formula (I) wherein: A is an antibody fragment thatspecifically binds to human cKIT; L_(B) is a linker; D is a cytotoxicagent; n is an integer from 1 to 10, and y is an integer from 1 to 10.2. The conjugate of claim 1, wherein n is 1, 2, 3, 4, 5, 6, 7 or
 8. 3.The conjugate of claim 1, wherein y is 1, 2, 3 or
 4. 4. The conjugate ofany one of claims 1 to 3, wherein each D is a cytotoxic agentindependently selected from an auristatin, an amanitin, a maytansinoidor a saporin.
 5. The conjugate of any one of claims 1 to 4, wherein eachD is a cytotoxic agent independently selected from an auristatin, anamanitin or a maytansinoid.
 6. The conjugate of any one of claims 1 to5, wherein each D is a cytotoxic agent independently selected from anauristatin or an amanitin.
 7. The conjugate of any one of claims 1 to 6,wherein each L_(B) is independently selected from a cleavable linker ora non-cleavable linker.
 8. The conjugate of any one of claims 1 to 7,wherein each L_(B) is a cleavable linker.
 9. The conjugate of any one ofclaims 1 to 7, wherein each L_(B) is a non-cleavable linker.
 10. Aconjugate having the structure of Formula (C):

wherein: A is an antibody fragment that specifically binds to humancKIT; y is an integer from 1 to 10; R² is C₁-C₆alkyl; L₂₀ is -L₁R⁴⁰; L₁is —((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-,—((CH₂)_(m)O)_(p)(CH₂)_(m)—, —(CH₂)_(m)—, —(CH₂)_(m)X(CH₂)_(m)—,—(CH₂)_(m)NHC(═O)(CH₂)_(m)—,—(CH₂)_(m)NHC(═O)(CH₂)_(m)C(═O)NH(CH₂)_(m)—,—((CH₂)_(m)O)_(p)(CH₂)_(m)NHC(═O)(CH₂)_(m),—((CH₂)_(m)O)_(p)CH₂)_(m)C(═O)NH(CH₂)_(m)—,X₃X₄C(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—, —X₃X₄C(═O)(CH₂)_(m)—,—X₃C(═O)(CH₂)_(n)NHC(═O)(CH₂)_(m)—,—X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—, —(CH₂)_(m)C(R₇)₂—,—(CH₂)_(m)C(R₇)₂SS(CH₂)_(m)NHC(═O)(CH₂)_(m)— or—(CH₂)_(m)X₃C(═O)(CH₂)_(m)NHC(═O)((CH₂)_(m)O)_(p)(CH₂)_(m)—; L₄ is—((CH₂)_(m);

 where the * indicates attachment point to L₄; X₂ is

 where the * indicates attachment point to L₄; X₃ is

X₄ is

R⁴⁰ is

 NR⁷C(═O)CH₂—, —NHC(═O)CH₂—, —S(═O)₂CH₂CH₂—, —(CH₂)₂S(═O)₂CH₂CH₂—,—NR⁷S(═O)₂CH₂CH₂, —NR⁷C(═O)CH₂CH₂—, —NH—, —C(═O)—, —NHC(═O)—,—CH₂NHCH₂CH₂—, —NHCH₂CH₂—, —S—,

each R⁷ is independently selected from H and C₁-C₆alkyl; each R¹⁰ isindependently selected from H, C₁-C₆alkyl, F, Cl, and —OH; each R¹¹ isindependently selected from H, C₁-C₆alkyl, F, Cl, —NH₂, —OCH₃, —OCH₂CH₃,—N(CH₃)₂, —CN, —NO₂ and —OH; each R¹² is independently selected from H,C₁₋₆alkyl, fluoro, benzyloxy substituted with —C(═O)OH, benzylsubstituted with —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OH andC₁₋₄alkyl substituted with —C(═O)OH; each R¹⁵ is independently selectedfrom H, —CH₃ and phenyl; each m is independently selected from 1, 2, 3,4, 5, 6, 7, 8, 9 and 10, and each p is independently selected from 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and
 14. 11. The conjugate of claim10, selected from


12. A conjugate having the structure of Formula (D):

wherein: A is an antibody fragment that specifically binds to humancKIT; y is an integer from 1 to 10; R¹ is

R² is C₁-C₆alkyl; L₃₀ is -L₅R⁴⁰; L₄ is —((CH₂)_(m); L₅ is—NHS(═O)₂(CH₂)_(m)X₁L₄, —NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-,—NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —NH((CH₂)_(m)O)_(p)(CH₂)_(m)—,—NH(CH₂)_(m)—, —NH(CH₂)_(m)X₁(CH₂)_(m)—, —NH(CH₂)_(m)NHC(═O)(CH₂)_(m)—,—NH(CH₂)_(m)NHC(═O)(CH₂)_(m)C(═O)NH(CH₂)_(m)—,—NH((CH₂)_(m)O)_(p)(CH₂)_(m)NHC(═O)(CH₂)_(m),—NH((CH₂)_(m)O)_(p)CH₂)_(m)C(═O)NH(CH₂)_(m)—, —NH(CH₂)_(n)C(R₇)₂—,—NH(CH₂)_(m)C(R₇)₂SS(CH₂)_(m)NHC(═O)(CH₂)_(m)— or—NH(CH₂)_(m)X₃C(═O)(CH₂)_(m)NHC(═O)((H₂)_(m)O)_(p)(CH₂)_(m)—; X₁ is

where the * indicates attachment point to L₄; X₂ is

where the * indicates attachment point to L₄; R⁴⁰ is

—NR⁷C(═O)CH₂—, —NHC(═O)CH₂—, —S(═O)₂CH₂CH₂—, —(CH₂)₂S(═O)₂CH₂CH₂—,—NR⁷S(═O)₂CH₂CH₂, —NR⁷C(═O)CH₂CH₂—, —NH—, —C(═O)—, —NHC(═O)—,—CH₂NHCH₂CH₂—, —NHCH₂CH₂—, —S—,

each R⁷ is independently selected from H and C₁-C₆alkyl; each R¹⁰ isindependently selected from H, C₁-C₆alkyl, F, Cl, and —OH; each R¹¹ isindependently selected from H, C₁-C₆alkyl, F, Cl, I, —NH₂, —OCH₃,—OCH₂CH₃, —N(CH₃)₂, —CN, —NO₂ and —OH; each R¹² is independentlyselected from H, C₁₋₆alkyl, fluoro, benzyloxy substituted with —C(═O)OH,benzyl substituted with —C(═O)OH, C₁₋₄alkoxy substituted with —C(═O)OHand C₁₋₄alkyl substituted with —C(═O)OH; each R¹⁵ is independentlyselected from H, —H₃ and phenyl; each m is independently selected from1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and each p is independently selectedfrom 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and
 14. 13. The conjugateof claim 12 selected from


14. A conjugate having the structure of Formula (E):

wherein: A is an antibody fragment that specifically binds to humancKIT; y is an integer from 1 to 10; X is S(═O), S(═O)₂ or S; R⁵ is H,—CH₃ or -CD₃; R⁶ is —NH₂ or —OH; L₄₀ is -L₆R⁴⁰; L₆ is is—((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-,-L₄NHC(═O)NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₁L₄-, -L₄NHC(═O)NH((CH₂)_(m)O)_(p)(CH₂)_(m)X₂L₄-, —((CH₂)_(m)O)_(p)(CH₂)_(m)—,—(CH₂)_(m)—, —(CH₂)_(m)X(CH₂)_(m)—, —(CH₂)_(m)NHC(═O)(CH₂)_(m)—,—(CH₂)_(m)NHC(═O)(CH₂)_(m)C(═O)NH(CH₂)_(m)—,—((CH₂)_(m)O)_(p)(CH₂)_(m)NHC(═O)(CH₂)_(m),—((CH₂)_(m)O)_(p)CH₂)_(m)C(═O)NH(CH₂)_(m)—, —(CH₂)_(m)C(R₇)₂— or—(CH₂)_(m)C(R₇)₂SS(CH₂)_(m)NHC(═O)(CH₂)_(m)—; L₄ is —((CH₂)_(m); X₁ is

 where the * indicates attachment point to L₄; X₂ is

 where the * indicates attachment point to L₄; R⁴⁰ is

 NR⁷C(═O)CH₂—, —NHC(═O)CH₂—, —S(═O)₂CH₂CH₂—, —(CH₂)₂S(═O)₂CH₂CH₂—,—NR⁷S(═O)₂CH₂CH₂, —NR⁷C(═O)CH₂CH₂—, —NH—, —C(═O)—, —NHC(═O)—,—CH₂NHCH₂CH₂—, —NHCH₂CH₂—, —S—,

each R⁷ is independently selected from H and C₁-C₆alkyl; each R¹⁰ isindependently selected from H, C₁-C₆alkyl, F, Cl, and OH; each R¹² isindependently selected from H, C₁₋₆alkyl, fluoro, benzyloxy substitutedwith —C(═O)OH, benzyl substituted with —C(═O)OH, C₁₋₄alkoxy substitutedwith —C(═O)OH and C₁₋₄alkyl substituted with —C(═O)OH; each R¹⁵ isindependently selected from H, —CH₃ and phenyl; each m is independentlyselected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and each p isindependently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13and
 14. 15. The conjugate of claim 14 selected from:


16. A conjugate selected from:

wherein: A is an antibody fragment that specifically binds to humancKIT, and y is an integer from 1 to
 10. 17. A conjugate selected from:

wherein: A is an antibody fragment that specifically binds to humancKIT, and y is an integer from 1 to
 10. 18. The conjugate of any one ofclaims 1 to 17, wherein the antibody fragment specifically binds to theextracellular domain of human cKIT (SEQ ID NO: 112).
 19. The conjugateof any one of claims 1 to 17, wherein the antibody fragment specificallybinds to an epitope in domains 1-3 of human cKIT (SEQ ID NO: 113). 20.The conjugate of any one of claims 1 to 19, wherein the antibodyfragment is a Fab or Fab′.
 21. The conjugate of any of claims 1 to 17 orclaim 20, wherein the antibody fragment is selected from any of thefollowing: (1) a Fab or Fab′ comprising (i) a heavy chain variableregion that comprises (a) a HCDR1 (Heavy Chain ComplementarityDetermining Region 1) of SEQ ID NO: 1, (b) a HCDR2 (Heavy ChainComplementarity Determining Region 2) of SEQ ID NO: 2, and (c) a HCDR3(Heavy Chain Complementarity Determining Region 3) of SEQ ID NO: 3; and(ii) a light chain variable region that comprises: (d) a LCDR1 (LightChain Complementarity Determining Region 1) of SEQ ID NO: 16, (e) aLCDR2 (Light Chain Complementarity Determining Region 2) of SEQ ID NO:17, and (f) a LCDR3 (Light Chain Complementarity Determining Region 3)of SEQ ID NO: 18; (2) a Fab or Fab′ comprising (i) a heavy chainvariable region that comprises (a) a HCDR1 of SEQ ID NO: 4, (b) a HCDR2of SEQ ID NO: 5, (c) a HCDR3 of SEQ ID NO: 3; and (ii) a light chainvariable region that comprises: (d) a LCDR1 of SEQ ID NO: 19, (e) aLCDR2 of SEQ ID NO: 20, and (f) a LCDR3 of SEQ ID NO: 21; (3) a Fab orFab′ comprising (i) a heavy chain variable region that comprises (a) aHCDR1 of SEQ ID NO: 6, (b) a HCDR2 of SEQ ID NO: 2, (c) a HCDR3 of SEQID NO: 3; and (ii) a light chain variable region that comprises: (d) aLCDR1 of SEQ ID NO: 16, (e) a LCDR2 of SEQ ID NO: 17, and (f) a LCDR3 ofSEQ ID NO: 18; (4) a Fab or Fab′ comprising (i) a heavy chain variableregion that comprises (a) a HCDR1 of SEQ ID NO: 7, (b) a HCDR2 of SEQ IDNO: 8, (c) a HCDR3 of SEQ ID NO: 9; and (ii) a light chain variableregion that comprises: (d) a LCDR1 of SEQ ID NO: 22, (e) a LCDR2 of SEQID NO: 20, and (f) a LCDR3 of SEQ ID NO: 18; (5) a Fab or Fab′comprising (i) a heavy chain variable region that comprises (a) a HCDR1of SEQ ID NO: 27, (b) a HCDR2 of SEQ ID NO: 28, (c) a HCDR3 of SEQ IDNO: 29; and (ii) a light chain variable region that comprises: (d) aLCDR1 of SEQ ID NO: 42, (e) a LCDR2 of SEQ ID NO: 17, and (f) a LCDR3 ofSEQ ID NO: 43; (6) a Fab or Fab′ comprising (i) a heavy chain variableregion that comprises (a) a HCDR1 of SEQ ID NO: 30, (b) a HCDR2 of SEQID NO: 31, (c) a HCDR3 of SEQ ID NO: 29; and (ii) a light chain variableregion that comprises: (d) a LCDR1 of SEQ ID NO: 44, (e) a LCDR2 of SEQID NO: 20, and (f) a LCDR3 of SEQ ID NO: 45; (7) a Fab or Fab′comprising (i) a heavy chain variable region that comprises (a) a HCDR1of SEQ ID NO: 32, (b) a HCDR2 of SEQ ID NO: 28, (c) a HCDR3 of SEQ IDNO: 29; and (ii) a light chain variable region that comprises: (d) aLCDR1 of SEQ ID NO: 42, (e) a LCDR2 of SEQ ID NO: 17, and (f) a LCDR3 ofSEQ ID NO: 43; (8) a Fab or Fab′ comprising (i) a heavy chain variableregion that comprises (a) a HCDR1 of SEQ ID NO: 33, (b) a HCDR2 of SEQID NO: 34, (c) a HCDR3 of SEQ ID NO: 35; and (ii) a light chain variableregion that comprises: (d) a LCDR1 of SEQ ID NO: 46, (e) a LCDR2 of SEQID NO: 20, and (f) a LCDR3 of SEQ ID NO: 43; (9) a Fab or Fab′comprising (i) a heavy chain variable region that comprises (a) a HCDR1of SEQ ID NO: 1, (b) a HCDR2 of SEQ ID NO: 51, (c) a HCDR3 of SEQ ID NO:3; and (ii) a light chain variable region that comprises: (d) a LCDR1 ofSEQ ID NO: 16, (e) a LCDR2 of SEQ ID NO: 17, and (f) a LCDR3 of SEQ IDNO: 18; (10) a Fab or Fab′ comprising (i) a heavy chain variable regionthat comprises (a) a HCDR1 of SEQ ID NO: 4, (b) a HCDR2 of SEQ ID NO:52, (c) a HCDR3 of SEQ ID NO: 3; and (ii) a light chain variable regionthat comprises: (d) a LCDR1 of SEQ ID NO: 19, (e) a LCDR2 of SEQ ID NO:20, and (f) a LCDR3 of SEQ ID NO: 21; (11) a Fab or Fab′ comprising (i)a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO:6, (b) a HCDR2 of SEQ ID NO: 51, (c) a HCDR3 of SEQ ID NO: 3; and (ii) alight chain variable region that comprises: (d) a LCDR1 of SEQ ID NO:16, (e) a LCDR2 of SEQ ID NO: 17, and (f) a LCDR3 of SEQ ID NO: 18; (12)a Fab or Fab′ comprising (i) a heavy chain variable region thatcomprises (a) a HCDR1 of SEQ ID NO: 7, (b) a HCDR2 of SEQ ID NO: 53, (c)a HCDR3 of SEQ ID NO: 9; and (ii) a light chain variable region thatcomprises: (d) a LCDR1 of SEQ ID NO: 22, (e) a LCDR2 of SEQ ID NO: 20,and (f) a LCDR3 of SEQ ID NO: 18; (13) a Fab or Fab′ comprising (i) aheavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 60,(b) a HCDR2 of SEQ ID NO: 61, (c) a HCDR3 of SEQ ID NO: 62; and (ii) alight chain variable region that comprises: (d) a LCDR1 of SEQ ID NO:75, (e) a LCDR2 of SEQ ID NO: 76, and (f) a LCDR3 of SEQ ID NO: 77; (14)a Fab or Fab′ comprising (i) a heavy chain variable region thatcomprises (a) a HCDR1 of SEQ ID NO: 63, (b) a HCDR2 of SEQ ID NO: 64,(c) a HCDR3 of SEQ ID NO: 62; and (ii) a light chain variable regionthat comprises: (d) a LCDR1 of SEQ ID NO: 78, (e) a LCDR2 of SEQ ID NO:79, and (f) a LCDR3 of SEQ ID NO: 80; (15) a Fab or Fab′ comprising (i)a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO:65, (b) a HCDR2 of SEQ ID NO: 61, (c) a HCDR3 of SEQ ID NO: 62; and (ii)a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO:75, (e) a LCDR2 of SEQ ID NO: 76, and (f) a LCDR3 of SEQ ID NO: 77; (16)a Fab or Fab′ comprising (i) a heavy chain variable region thatcomprises (a) a HCDR1 of SEQ ID NO: 66, (b) a HCDR2 of SEQ ID NO: 67,(c) a HCDR3 of SEQ ID NO: 68; and (ii) a light chain variable regionthat comprises: (d) a LCDR1 of SEQ ID NO: 81, (e) a LCDR2 of SEQ ID NO:79, and (f) a LCDR3 of SEQ ID NO: 77; (17) a Fab or Fab′ comprising (i)a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO:86, (b) a HCDR2 of SEQ ID NO: 87, (c) a HCDR3 of SEQ ID NO: 88; and (ii)a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO:101, (e) a LCDR2 of SEQ ID NO: 102, and (f) a LCDR3 of SEQ ID NO: 103;(18) a Fab or Fab′ comprising (i) a heavy chain variable region thatcomprises (a) a HCDR1 of SEQ ID NO: 89, (b) a HCDR2 of SEQ ID NO: 90,(c) a HCDR3 of SEQ ID NO: 88; and (ii) a light chain variable regionthat comprises: (d) a LCDR1 of SEQ ID NO: 104, (e) a LCDR2 of SEQ ID NO:105, and (f) a LCDR3 of SEQ ID NO: 106; (19) a Fab or Fab′ comprising(i) a heavy chain variable region that comprises (a) a HCDR1 of SEQ IDNO: 91, (b) a HCDR2 of SEQ ID NO: 87, (c) a HCDR3 of SEQ ID NO: 88; and(ii) a light chain variable region that comprises: (d) a LCDR1 of SEQ IDNO: 101, (e) a LCDR2 of SEQ ID NO: 102, and (f) a LCDR3 of SEQ ID NO:103; (20) a Fab or Fab′ comprising (i) a heavy chain variable regionthat comprises (a) a HCDR1 of SEQ ID NO: 92, (b) a HCDR2 of SEQ ID NO:93, (c) a HCDR3 of SEQ ID NO: 94; and (ii) a light chain variable regionthat comprises: (d) a LCDR1 of SEQ ID NO: 107, (e) a LCDR2 of SEQ ID NO:105, and (f) a LCDR3 of SEQ ID NO: 103; (21) a Fab or Fab′ comprising aheavy chain variable region (VH) that comprises SEQ ID NO: 10, and alight chain variable region (VL) that comprises SEQ ID NO: 23; (22) aFab or Fab′ comprising a VH that comprises SEQ ID NO: 36, and a VL thatcomprises SEQ ID NO: 47; (23) a Fab or Fab′ comprising a VH thatcomprises SEQ ID NO: 54, and a VL that comprises SEQ ID NO: 23; (24) aFab or Fab′ comprising a VH that comprises SEQ ID NO: 69, and a VL thatcomprises SEQ ID NO: 82; (25) a Fab or Fab′ comprising a VH thatcomprises SEQ ID NO: 95, and a VL that comprises SEQ ID NO: 108; (26) aFab′ comprising a heavy chain that that comprises SEQ ID NO: 14, and alight chain that comprises SEQ ID NO: 25; (27) a Fab′ comprising a heavychain that that comprises SEQ ID NO: 40, and a light chain thatcomprises SEQ ID NO: 49; (28) a Fab′ comprising a heavy chain that thatcomprises SEQ ID NO: 58, and a light chain that comprises SEQ ID NO: 25;(29) a Fab′ comprising a heavy chain that that comprises SEQ ID NO: 73,and a light chain that comprises SEQ ID NO: 84; (30) a Fab′ comprising aheavy chain that that comprises SEQ ID NO: 99, and a light chain thatcomprises SEQ ID NO:110; (31) a Fab comprising a heavy chain thatcomprises SEQ ID NO: 118, and a light chain that comprises SEQ ID NO:122; (32) a Fab comprising a heavy chain that comprises SEQ ID NO: 118,and a light chain that comprises SEQ ID NO: 123; (33) a Fab comprising aheavy chain that comprises SEQ ID NO: 124, and a light chain thatcomprises SEQ ID NO: 128; (34) a Fab comprising a heavy chain thatcomprises SEQ ID NO: 124, and a light chain that comprises SEQ ID NO:129; (35) a Fab comprising a heavy chain that comprises SEQ ID NO: 130,and a light chain that comprises SEQ ID NO: 134; (36) a Fab comprising aheavy chain that comprises SEQ ID NO: 130, and a light chain thatcomprises SEQ ID NO: 135; (37) a Fab comprising a heavy chain thatcomprises SEQ ID NO: 136, and a light chain that comprises SEQ ID NO:140; (38) a Fab comprising a heavy chain that comprises SEQ ID NO: 141,and a light chain that comprises SEQ ID NO: 145; (39) a Fab comprising aheavy chain that comprises an amino acid sequence selected from SEQ IDNO: 119, 120 or 121, and a light chain comprising the amino acidsequence of SEQ ID NO: 25; (40) a Fab comprising a heavy chain thatcomprises an amino acid sequence selected from SEQ ID NO: 125, 126, or127, and a light chain comprising the amino acid sequence of SEQ ID NO:49; (41) a Fab comprising a heavy chain that comprises an amino acidsequence selected from SEQ ID NO: 131, 132, or 133, and a light chaincomprising the amino acid sequence of SEQ ID NO: 25; (42) a Fabcomprising a heavy chain that comprises an amino acid sequence selectedfrom SEQ ID NO: 137, 138, or 139, and a light chain comprising the aminoacid sequence of SEQ ID NO: 84; or (43) a Fab comprising a heavy chainthat comprises an amino acid sequence selected from SEQ ID NO: 142, 143,or 144, and a light chain comprising the amino acid sequence of SEQ IDNO:
 110. 22. The conjugate of any one of claims 1 to 21, wherein theantibody fragment is a human or humanized Fab or Fab′.
 23. The conjugateof any one of claims 1 to 9, wherein the antibody fragment is a Fab′ andthe linker (L_(B)) is attached to a native cysteine residue in the hingeregion of the Fab′.
 24. The conjugate of any one of claims 1 to 9,wherein the antibody fragment comprises at least one non-native cysteineintroduced into a constant region, and the linker (L_(B)) is attached tothe non-native cysteine.
 25. The conjugate of any one of claims 10 to 11or claims 14-15, wherein the antibody fragment is a Fab′ and L₂₀ isattached to a native cysteine residue in the hinge region of the Fab′.26. The conjugate of any one of claims 10 to 11 or claims 14-15, whereinthe antibody fragment comprises at least one non-native cysteineintroduced into a constant region, and L₂₀ is attached to the non-nativecysteine.
 27. The conjugate of any one of claims 12 to 13, wherein theantibody fragment is a Fab′ and L₃₀ is attached to a native cysteineresidue in the hinge region of the Fab′.
 28. The conjugate of any one ofclaims 12 to 13, wherein the antibody fragment comprises at least onenon-native cysteine introduced into a constant region, and L₃₀ isattached to the non-native cysteine.
 29. The conjugate of any one ofclaims 24, 26 or 28, wherein the antibody fragment comprises cysteine atone or more of the following positions (all positions by EU numbering):(a) position 152 of the heavy chain, (b) position 114 or 165 of thekappa light chain, or (c) position 143 of the lambda light chain. 30.The conjugate of any one of claims 1 to 29, wherein the half-life of theconjugate is less than about 24-48 hours.
 31. The conjugate of any oneof claims 1 to 30, wherein the conjugate does not induce mast celldegranulation.
 32. A pharmaceutical composition comprising the conjugateof any one of claims 1 to 31 and a pharmaceutically acceptable carrier.33. The pharmaceutical composition of claim 32 further comprisinganother therapeutic agent.
 34. The pharmaceutical composition of claim32, wherein the composition is a lyophilisate.
 35. A method of ablatinghematopoietic stem cells in a patient in need thereof, the methodcomprising administering to the patient an effective amount of theconjugate of any of claims 1-31, or the pharmaceutical composition ofclaim 32 or
 33. 36. The method of claim 35, wherein the patient is ahematopoietic stem cell transplantation recipient.
 37. The method ofclaim 36, wherein the method is performed before hematopoietic stem celltransplantation to the patient.
 38. A method of conditioning ahematopoietic stem cell transplantation patient, the method comprising:administering to the patient an effective amount of the conjugate of anyof claims 1-31, or the pharmaceutical composition of claim 32 or 33, andallowing a sufficient period of time for the conjugates to clear fromthe patient's circulation before performing hematopoietic stem celltransplantation to the patient.
 39. The method of any of claims 35-38,wherein the patient has an inherited immunodeficient disease, anautoimmune disorder, a hematopoietic disorder, or an inborn error ofmetabolism.
 40. The method of claim 39, wherein the hematopoieticdisorder is selected from: Acute myeloid leukemia (AML), Acutelymphoblastic leukemia (ALL), acute monocytic leukemia (AMoL), Chronicmyeloid leukemia (CML), Chronic lymphocytic leukemia (CLL),Myeloproliferative disorders, Myelodysplastic syndromes, Multiplemyeloma, Non-Hodgkin lymphoma, Hodgkin disease, Aplastic anemia, Purered cell aplasia, Paroxysmal nocturnal hemoglobinuria, Fanconi anemi,Thalassemia major, Sickle cell anemia, Severe combined immunodeficiency,Wiskott-Aldrich syndrome, Hemophagocytic lymphohistiocytosis.
 41. Themethod of claim 39, wherein the inborn error of metabolism is selectedfrom mucopolysaccharidosis, Gaucher disease, metachromaticleukodystrophies, or adrenoleukodystrophies.
 42. The method of any ofclaims 35-38, wherein the patient has a non-malignant disease orcondition selected from Severe aplastic anemia (SAA), Wiskott AldrichSyndrome, Hurlers Syndrome, FHL, CGD, Kostmanns syndrome, Severeimmunodeficiency syndrome (SCID), other autoimmune disorders such asSLE, Multiple sclerosis, IBD, Crolms Disease, Sjogrens syndrome,vasculitis, Lupus, Myasthenia Gravis, Wegeners disease, inborn errors ofmetabolism and/or other immunodeficiencies.
 43. The method of any ofclaims 35-38, wherein the patient has a malignant disease or conditionselected from myelodysplastic syndromes (MDS), acute lymphoblasticleukemia (ALL), acute myelogenous leukemia (AML), acute monocyticleukemia (AMoL), chronic lymphocytic leukemia (CLL), chronic myelogenousleukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocyticleukemia (T-PLL), large granular lymphocytic leukemia, adult T-cellleukemia, Precursor T-cell leukemia/lymphoma, Burkitt lymphoma,follicular lymphoma, diffuse large B cell lymphoma, mantle celllymphoma, B-cell chronic lymphocytic leukemia/lymphoma, MALT lymphoma,Mycosis fungoides, Peripheral T-cell lymphoma not otherwise specified,Nodular sclerosis form of Hodgkin lymphoma Mixed-cellularity subtype ofHodgkin lymphoma.
 44. Use of the conjugate of any of claims 1-31, or thepharmaceutical composition of claim 31 or 32, for ablating hematopoieticstem cells in a patient in need thereof.
 45. Use of the conjugate of anyof claims 1-31, or the pharmaceutical composition of claim 31 or 32, inthe manufacture of a medicament for ablating hematopoietic stem cells ina patient in need thereof.
 46. An antibody or antibody fragment thatspecifically binds to human cKIT, wherein the antibody or antibodyfragment is selected from any of the following: (1) an antibody orantibody fragment comprising (i) a heavy chain variable region thatcomprises (a) a HCDR1 (Heavy Chain Complementarity Determining Region 1)of SEQ ID NO: 1, (b) a HCDR2 (Heavy Chain Complementarity DeterminingRegion 2) of SEQ ID NO: 2, and (c) a HCDR3 (Heavy Chain ComplementarityDetermining Region 3) of SEQ ID NO: 3; and (ii) a light chain variableregion that comprises: (d) a LCDR1 (Light Chain ComplementarityDetermining Region 1) of SEQ ID NO: 16, (e) a LCDR2 (Light ChainComplementarity Determining Region 2) of SEQ ID NO: 17, and (f) a LCDR3(Light Chain Complementarity Determining Region 3) of SEQ ID NO: 18; (2)an antibody or antibody fragment comprising (i) a heavy chain variableregion that comprises (a) a HCDR1 of SEQ ID NO: 4, (b) a HCDR2 of SEQ IDNO: 5, (c) a HCDR3 of SEQ ID NO: 3; and (ii) a light chain variableregion that comprises: (d) a LCDR1 of SEQ ID NO: 19, (e) a LCDR2 of SEQID NO: 20, and (f) a LCDR3 of SEQ ID NO: 21; (3) an antibody or antibodyfragment comprising (i) a heavy chain variable region that comprises (a)a HCDR1 of SEQ ID NO: 6, (b) a HCDR2 of SEQ ID NO: 2, (c) a HCDR3 of SEQID NO: 3; and (ii) a light chain variable region that comprises: (d) aLCDR1 of SEQ ID NO: 16, (e) a LCDR2 of SEQ ID NO: 17, and (f) a LCDR3 ofSEQ ID NO: 18; (4) an antibody or antibody fragment comprising (i) aheavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 7,(b) a HCDR2 of SEQ ID NO: 8, (c) a HCDR3 of SEQ ID NO: 9; and (ii) alight chain variable region that comprises: (d) a LCDR1 of SEQ ID NO:22, (e) a LCDR2 of SEQ ID NO: 20, and (f) a LCDR3 of SEQ ID NO: 18; (5)an antibody or antibody fragment comprising (i) a heavy chain variableregion that comprises (a) a HCDR1 of SEQ ID NO: 27, (b) a HCDR2 of SEQID NO: 28, (c) a HCDR3 of SEQ ID NO: 29; and (ii) a light chain variableregion that comprises: (d) a LCDR1 of SEQ ID NO: 42, (e) a LCDR2 of SEQID NO: 17, and (f) a LCDR3 of SEQ ID NO: 43; (6) an antibody or antibodyfragment comprising (i) a heavy chain variable region that comprises (a)a HCDR1 of SEQ ID NO: 30, (b) a HCDR2 of SEQ ID NO: 31, (c) a HCDR3 ofSEQ ID NO: 29; and (ii) a light chain variable region that comprises:(d) a LCDR1 of SEQ ID NO: 44, (e) a LCDR2 of SEQ ID NO: 20, and (f) aLCDR3 of SEQ ID NO: 45; (7) an antibody or antibody fragment comprising(i) a heavy chain variable region that comprises (a) a HCDR1 of SEQ IDNO: 32, (b) a HCDR2 of SEQ ID NO: 28, (c) a HCDR3 of SEQ ID NO: 29; and(ii) a light chain variable region that comprises: (d) a LCDR1 of SEQ IDNO: 42, (e) a LCDR2 of SEQ ID NO: 17, and (f) a LCDR3 of SEQ ID NO: 43;(8) an antibody or antibody fragment comprising (i) a heavy chainvariable region that comprises (a) a HCDR1 of SEQ ID NO: 33, (b) a HCDR2of SEQ ID NO: 34, (c) a HCDR3 of SEQ ID NO: 35; and (ii) a light chainvariable region that comprises: (d) a LCDR1 of SEQ ID NO: 46, (e) aLCDR2 of SEQ ID NO: 20, and (f) a LCDR3 of SEQ ID NO: 43; (9) anantibody or antibody fragment comprising (i) a heavy chain variableregion that comprises (a) a HCDR1 of SEQ ID NO: 60, (b) a HCDR2 of SEQID NO: 61, (c) a HCDR3 of SEQ ID NO: 62; and (ii) a light chain variableregion that comprises: (d) a LCDR1 of SEQ ID NO: 75, (e) a LCDR2 of SEQID NO: 76, and (f) a LCDR3 of SEQ ID NO: 77; (10) an antibody orantibody fragment comprising (i) a heavy chain variable region thatcomprises (a) a HCDR1 of SEQ ID NO: 63, (b) a HCDR2 of SEQ ID NO: 64,(c) a HCDR3 of SEQ ID NO: 62; and (ii) a light chain variable regionthat comprises: (d) a LCDR1 of SEQ ID NO: 78, (e) a LCDR2 of SEQ ID NO:79, and (f) a LCDR3 of SEQ ID NO: 80; (11) an antibody or antibodyfragment comprising (i) a heavy chain variable region that comprises (a)a HCDR1 of SEQ ID NO: 65, (b) a HCDR2 of SEQ ID NO: 61, (c) a HCDR3 ofSEQ ID NO: 62; and (ii) a light chain variable region that comprises:(d) a LCDR1 of SEQ ID NO: 75, (e) a LCDR2 of SEQ ID NO: 76, and (f) aLCDR3 of SEQ ID NO: 77; (12) an antibody or antibody fragment comprising(i) a heavy chain variable region that comprises (a) a HCDR1 of SEQ IDNO: 66, (b) a HCDR2 of SEQ ID NO: 67, (c) a HCDR3 of SEQ ID NO: 68; and(ii) a light chain variable region that comprises: (d) a LCDR1 of SEQ IDNO: 81, (e) a LCDR2 of SEQ ID NO: 79, and (f) a LCDR3 of SEQ ID NO: 77;(13) an antibody or antibody fragment comprising (i) a heavy chainvariable region that comprises (a) a HCDR1 of SEQ ID NO: 86, (b) a HCDR2of SEQ ID NO: 87, (c) a HCDR3 of SEQ ID NO: 88; and (ii) a light chainvariable region that comprises: (d) a LCDR1 of SEQ ID NO: 101, (e) aLCDR2 of SEQ ID NO: 102, and (f) a LCDR3 of SEQ ID NO: 103; (14) anantibody or antibody fragment comprising (i) a heavy chain variableregion that comprises (a) a HCDR1 of SEQ ID NO: 89, (b) a HCDR2 of SEQID NO: 90, (c) a HCDR3 of SEQ ID NO: 88; and (ii) a light chain variableregion that comprises: (d) a LCDR1 of SEQ ID NO: 104, (e) a LCDR2 of SEQID NO: 105, and (f) a LCDR3 of SEQ ID NO: 106; (15) an antibody orantibody fragment comprising (i) a heavy chain variable region thatcomprises (a) a HCDR1 of SEQ ID NO: 91, (b) a HCDR2 of SEQ ID NO: 87,(c) a HCDR3 of SEQ ID NO: 88; and (ii) a light chain variable regionthat comprises: (d) a LCDR1 of SEQ ID NO: 101, (e) a LCDR2 of SEQ ID NO:102, and (f) a LCDR3 of SEQ ID NO: 103; (16) an antibody or antibodyfragment comprising (i) a heavy chain variable region that comprises (a)a HCDR1 of SEQ ID NO: 92, (b) a HCDR2 of SEQ ID NO: 93, (c) a HCDR3 ofSEQ ID NO: 94; and (ii) a light chain variable region that comprises:(d) a LCDR1 of SEQ ID NO: 107, (e) a LCDR2 of SEQ ID NO: 105, and (f) aLCDR3 of SEQ ID NO: 103; (17) an antibody or antibody fragmentcomprising a heavy chain variable region (VH) that comprises SEQ ID NO:10, and a light chain variable region (VL) that comprises SEQ ID NO: 23;(18) an antibody or antibody fragment comprising a VH that comprises SEQID NO: 36, and a VL that comprises SEQ ID NO: 47; (19) an antibody orantibody fragment comprising a VH that comprises SEQ ID NO: 69, and a VLthat comprises SEQ ID NO: 82; (20) an antibody or antibody fragmentcomprising a VH that comprises SEQ ID NO: 95, and a VL that comprisesSEQ ID NO: 108; (21) an antibody or antibody fragment comprising a heavychain that that comprises SEQ ID NO: 14, and a light chain thatcomprises SEQ ID NO: 25; (22) an antibody or antibody fragmentcomprising a heavy chain that that comprises SEQ ID NO: 40, and a lightchain that comprises SEQ ID NO: 49; (23) an antibody or antibodyfragment comprising a heavy chain that that comprises SEQ ID NO: 73, anda light chain that comprises SEQ ID NO: 84; (24) an antibody or antibodyfragment comprising a heavy chain that that comprises SEQ ID NO: 99, anda light chain that comprises SEQ ID NO: 110; (25) an antibody orantibody fragment comprising a heavy chain that comprises SEQ ID NO:118, and a light chain that comprises SEQ ID NO: 122; (26) an antibodyor antibody fragment comprising a heavy chain that comprises SEQ ID NO:118, and a light chain that comprises SEQ ID NO: 123; (27) an antibodyor antibody fragment comprising a heavy chain that comprises SEQ ID NO:124, and a light chain that comprises SEQ ID NO: 128; (28) an antibodyor antibody fragment comprising a heavy chain that comprises SEQ ID NO:124, and a light chain that comprises SEQ ID NO: 129; (29) an antibodyor antibody fragment comprising a heavy chain that comprises SEQ ID NO:130, and a light chain that comprises SEQ ID NO: 134; (30) an antibodyor antibody fragment comprising a heavy chain that comprises SEQ ID NO:130, and a light chain that comprises SEQ ID NO: 135; (31) an antibodyor antibody fragment comprising a heavy chain that comprises SEQ ID NO:136, and a light chain that comprises SEQ ID NO: 140; (32) an antibodyor antibody fragment comprising a heavy chain that comprises SEQ ID NO:141, and a light chain that comprises SEQ ID NO: 145; (33) an antibodycomprising a heavy chain that that comprises SEQ ID NO: 12, and a lightchain that comprises SEQ ID NO: 25; (34) an antibody comprising a heavychain that that comprises SEQ ID NO: 38, and a light chain thatcomprises SEQ ID NO: 49; (35) an antibody comprising a heavy chain thatthat comprises SEQ ID NO: 71, and a light chain that comprises SEQ IDNO: 84; or (36) an antibody comprising a heavy chain that that comprisesSEQ ID NO: 97, and a light chain that comprises SEQ ID NO:
 110. 47. Anucleic acid encoding an antibody or antibody fragment of claim
 46. 48.A vector comprising the nucleic acid of claim
 47. 49. A host cellcomprising the vector of claim
 48. 50. A process for producing anantibody or antibody fragment, the process comprising cultivating thehost cell of claim 49, and recovering the antibody or antibody fragmentfrom the culture.